Processes for the digestion of colanic acid

ABSTRACT

The present disclosure generally relates to processes employing polypeptides having colanic acid-degrading activity. The processes generally involve contacting a biological material with a polypeptide capable of digesting colanic acid. Additional process steps, such as chromatographic separation steps, are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims the benefit of U.S. Provisional ApplicationSer. No. 61/125,916, filed on Apr. 30, 2008; and Ser. No. 61/125,923,filed on Apr. 30, 2008, each of which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present disclosure generally relates to processes employingpolypeptides having colanic acid-degrading activity. The processesgenerally involve contacting a biological material with a polypeptidecapable of digesting colanic acid. Additional process steps, such aschromatographic separation steps, are also described.

BACKGROUND

A key step for any application in which nucleic acid is introduced intoan organism is the need to produce highly purified, often pharmaceuticalgrade, nucleic acid. Such purified nucleic acid must meet drug qualitystandards of safety, potency and efficacy. In addition, it is desirableto have a scaleable process that can be used to produce multi-gramquantities of DNA. Thus, it is desirable to have a process for producinghighly pure nucleic acid that does not require toxic chemicals,mutagens, organic solvents, or other reagents that would compromise thesafety or efficacy of the resulting nucleic acid, or make scale-updifficult or impractical. It is also desirable to prepare nucleic acidsfree from contaminating endotoxins, which if administered to a patientcould elicit a toxic response. Removal of contaminating endotoxins isparticularly important, for example, where plasmid DNA is purified fromgram negative (−) bacterial sources that have high levels of endotoxinsand colanic acid as an integral component of the outer cell membrane.

Plasmids are self-replicating genetic elements that reside and multiplyin host bacteria. Basically, all molecular genetic methods involving themanipulation of specific DNA fragments utilize plasmid DNA to producelarge amounts of the specific DNA fragment (or protein/RNA derived fromsaid fragment).

The choice of bacterial hosts, or sources of the plasmids, generallyreflects a historical perspective. Stanley Cohen and Herb Bayer choseEscherichia coli (E. coli) strain K-12 for their groundbreakingmolecular genetic experiments in the early 1970s because it was easy togrow and amenable to metabolic studies. These same properties also madeE. coli K-12 the primary microorganism for bacterial geneticists tostudy. Molecular geneticists now use this same strain of E. coli forroutine procedures because it turned out to be an extremely good hostfor a variety of molecular genetic applications. Moreover, during thepast 25 years, E. coli K-12 has proved to be an innocuous biologicalhost for the propagation of recombinant DNA molecules. The attenuated E.coli K-12 strain does not thrive outside of the laboratory environmentand it is unable to compete against the more genetically robust E. coliserotypes normally found in the human intestine.

Among other techniques, currently available methods for separation andpurification of plasmid DNA utilize ion exchange chromatography (Duarteet al., Journal of Chromatography A, 606 (1998), 31-45) and sizeexclusion chromatography (Prazeres, D. M., Biotechnology Techniques Vol.1, No. 6, June 1997, p 417-420), coupled with the use of additives suchas polyethylene glycol (PEG), detergents, and other components such ashexamine cobalt, spermidine, and polyvinylpyrollidone (PVP). Additionalmethods of separating DNA from contaminants rely on size-exclusionchromatography, which involves separation of the nucleic acid fromendotoxins and other contaminants based on the small difference in size.These methods are generally acceptable, but may be unable to provide anefficient and cost effective separation of nucleic acids (e.g., DNA,including supercoiled and/or nicked (or relaxed)) at the desired levelof purity.

Also, plasmid DNA preparations, which are produced from bacterialpreparations and often contain a mixture of relaxed and supercoiledplasmid DNA, frequently require endotoxin removal, as required by theFDA, as endotoxins produced by many bacterial hosts are known to causeinflammatory reactions, such as fever or sepsis, or in some cases death,in the host receiving the plasmid DNA. These endotoxins are generallylipopolysaccharides, or fragments thereof, that are components of theouter membrane of gram negative (−) bacteria, and are present in the DNApreparation of the host cells and host cell membranes or macromolecules.Hence, removal of endotoxins can be a key step in the purification ofplasmid DNA for therapeutic or prophylactic use. Endotoxin removal fromplasmid DNA solutions primarily uses the negatively charged structure ofthe endotoxins. Plasmid DNA, however, also is negatively charged andthus separation is frequently achieved with anion exchange resins whichbind both these molecules and, under certain conditions, preferentiallyelute plasmid DNA while binding the endotoxins. Such a separationresults in only partial removal as significant amounts of endotoxinselute with the plasmid DNA and/or a very poor recovery of plasmid DNA isachieved.

Small- and large-scale isolation and purification of plasmid DNA fromsmall or large volume microbial fermentations thus requires thedevelopment of an improved plasmid preparation process. It is alsodesirable for plasmid-based research and therapy, that the nucleic acidscan be separated and purified keeping the same structure in areproducible manner, and in order to avoid the adverse effect ofimpurities on mammalian body, the nucleic acids are required to havebeen separated and purified up to high purity.

Plasmid DNA used for gene therapy is typically isolated from E. coliK-12. Endotoxins, also known as lipopolysaccharides (LPS), are known tobe prominent cell membrane components of gram-negative bacteria such asE. coli. In fact, some reports suggest that the lipid portion of theouter membrane of E. coli is completely composed of endotoxin molecules.(Qiagen Plasmid Purification Handbook, July 1999).

LPS contains a hydrophobic lipid A moiety, a complex array of sugarresidues and negatively charged phosphate groups. The lipid A moiety ofLPS has demonstrated endotoxin activity and elicits a strong,potentially life-threatening inflammatory response in mammals. Thisinflammatory response is characterized by fever, decreased bloodpressure, local inflammation, and septic shock. Lipid A induces thisresponse by binding to serum lipopolysaccharide-binding protein (LBP)and triggering signaling through the CD14 receptor expressed onmonocytes, endothelial cells, and polymorphonuclear leukocytes (Ingalls,R. R. et al. 1998. J. Immunol. 161:5413-5420). Endotoxin is extremelylethal when injected into mice, causing death within an hour ofinjection. Endotoxin is also known to drastically reduce transfectionefficiencies in cells (Weber, M., et al. 1995. Biotechniques19:930-940). Thus, the importance of using endotoxin-free plasmid DNAfor gene therapy applications has long been emphasized.

A number of scientists have worked to remove LPS and other endotoxinsfrom DNA samples in an effort to reduce the toxicity of DNA samples usedin gene therapy. However, recent evidence has indicated that DNA sampleswith negligible amounts of LPS are still toxic when administered insignificant quantities. Thus, additional toxic components of DNA samplesmust be identified and removed to ensure the safety of DNA preparationsused clinically.

The chemical structure and properties of endotoxin molecules and theirtendency to form micellar structures initially led to the copurificationof LPS and plasmid DNA. For example, DNA is often copurified with LPS inCsCl ultracentrifugation procedures because the LPS and the plasmid DNAhave a similar density in CsCl. In addition, micellar LPS separates onsize exclusion resins with large DNA molecules. Likewise, the negativecharges present on LPS molecules interacts with anion-exchange resins ina manner that leads to their copurification with DNA on anion-exchangeresins.

Cell wall polysaccharides have been reported to contaminate DNA isolatedfrom a variety of sources including bacteria, yeast, plants, blue-greenalgae, protozoa, fungi, insects, and mammals (Edelman, M. 1975. Anal.Biochem. 65:293-297; Do, N. and Adams, R. P. 1991. Biotechniques10:162-166; Chan, J. W. and Goodwin, P. H. 1995. Biotechniques18:419-422).

Plant polysaccharides that contaminate plant genomic DNA are reported toinhibit both restriction endonuclease treatments and the polymerasechain reaction (Robbins, M. et al. 1995. Benchmarks 18: 419-422).Furthermore, polysaccharides purified from the slime Physarumpolycephalum have been reported to inhibit DNA polymerase activity(Shioda, M. and K. Murakami-Murofushi. 1987. Biochem. Biophys. Res.Commun. 146:61-66) and the acid polysaccharides from sea urchin embryosare known to inhibit RNA polymerase activity (Aoki, Y. and H. Koshihara.1972. Biochim. Biophys. Acta 272:33-43).

There are several methods for purifying plasmid DNA described in theliterature, but these methods generally only remove a portion of thepolysaccharides, if at all. For example, the Lipid A purificationmethods are based on the hydrophobic properties of Lipid A. Thus, thesemethods remove Lipid A and the polysaccharides that are covalentlylinked to Lipid A. However, since only a small fraction of the capsularpolysaccharides of E. coli are covalently linked to Lipid A only a fewof them are removed during the standard preparation and purificationprocedures of plasmid DNA (Jann, B. and K. Jann. 1990. Curr. Top.Microbiol. Immunol. 150:19-42; Wicken, A. J. 1985. In: BacterialAdhesion, D. M. Pletcher (ed.), Plenum Press: New York, pp. 45-70). Someof the E. coli capsular polysaccharides have phosphatidic acid as alipid moiety; however, the phosphatidic acid is typically hydrolyzedduring the standard plasmid isolation procedures. Thus, thesepolysaccharides are not removed from DNA by the currently used methodsthat deplete endotoxin based on hydrophobicity (i.e., the presence ofLipid A binding).

Several methods have been developed to reduce levels ofendotoxin-positive LPS in DNA isolated from E. coli (Neudecker, F. andS. Grimm. 2000. Biotechniques 28:107-110), including severalcommercially available kits (Qiagen, Inc., Valencia, Calif.; SigmaChemical Co., Inc., St. Louis, Mo.). DNA purified using the Qiagen kitis generally considered to be the “gold standard” of clean plasmid DNA.Not only is the Qiagen kit designed to remove LPS, but it also includesRNase to digest the RNA in plasmid DNA preparations. In fact, most ofthe DNA purification methods include an RNase digestion step. However,one is limited to the amount of RNase that can be added to the plasmidDNA, since high quantities of RNase will begin to digest DNA.

It is difficult to separate polysaccharides from DNA using currentstandard purification procedures. Both DNA and polysaccharides areprecipitated by organic solvents such as ethanol and polyethylene glycol(PEG). Since polysaccharides are anionic, the polysaccharides co-purifywith DNA using anion exchange resins. Furthermore, the high molecularweight polysaccharides and plasmid DNA have a similar density in CsCl.

Affinity chromatography has been proposed for removal of polysaccharidecontaminants from DNA. For example, an early paper reported purificationof DNA from a variety of sources, including plants, insects, fungi, andalgae using affinity chromatography where deproteinized DNA fractionsare passed through a column of concanavalin A linked to Sepharose(Edelman, M. 1975. Anal. Biochem. 65:293-29). Unfortunately, E. colipolysaccharides generally do not contain the sugars that bind toconcanavalin A. Similarly, lectin affinity chromatography has beenreported to be useful for removing polysaccharide contaminants from DNAisolated from fungi and plants (Do, N. and R. P. Adams. 1991.Biotechniques 10:162-166); but the sugars recognized by lectin are notpresent in most polysaccharides from organisms such as E. coli.

A salt wash of gram-negative bacterial pellets has also been proposed asa method of purifying bacterial genomic DNA (Cahn, J. W. and P. H.Goodwin. 1995. Biotechniques 18:519-422). Salt washing was suggested asa way to improve purification of DNA because of the interference thepolysaccharides present in DNA caused with restriction enzyme digestion.None of these methods, however, successfully removed all polysaccharidesfound in plasmid DNA.

WO 95/20594 and U.S. Pat. No. 5,969,129 describe a method for batchpurification of genomic DNA, from corn and other plants. Thispurification process used polymer gels containing boronate groups toisolate DNA from DNA/polysaccharide mixtures.

Although, the entire emphasis of clinicians in preparing “clean” DNA forclinical use has centered on the removal of LPS, recent reports indicatethat “LPS-free” DNA still exhibits toxicity in high dosages. Forexample, scientists have observed toxicity leading to the death of micefollowing intravenous injection of DNA-liposome complexes containing50-300 mg of DNA and reduced quantities of LPS, whereas the injection ofequal concentrations of liposomes has no toxic effect. It has also beenobserved that gene expression is reduced after transfection using DNAwith reduced quantities of LPS. Recent reports further suggest thatinflammation and significant immune responses are produced after theintramuscular injection of supposedly pure DNA (Fields, P. A. et al.2000. Molec. Therap. 1:225-235). Therefore, even DNA that is thought tobe pure, of clinical grade, and with low levels of LPS, produces toxicresponses in animals.

SUMMARY OF THE INVENTION

Among the various aspects of the present invention is the provisionprocesses for digesting or degrading colanic acid.

Briefly, therefore, the present invention is directed to a process fordigesting colanic acid in a biological material, the process comprisingcontacting the biological material with a polypeptide capable ofdigesting colanic acid. In one embodiment, the biological material isselected from a crude bacterial lysate, a partially purified bacteriallysate, and an aqueous solution containing extracted bacterial nucleicacid. In another embodiment, the biological material is a bacterialslime. In another embodiment, the biological material is a biofilm.

Another aspect of the invention is directed to a process for the removalof endotoxin from an aqueous composition containing bacterialmacromolecules, the process comprising digesting colanic acid in theaqueous composition and thereafter combining the aqueous compositionwith a chromatographic material to separate endotoxin from the bacterialmacromolecule. In certain embodiments, the aqueous composition isderived from a bacterial lysate.

Other aspects and features will be in part apparent and in part pointedout hereinafter.

The foregoing has outlined rather broadly several aspects of the presentinvention in order that the detailed description of the invention thatfollows may be better understood. Additional features and advantages ofthe invention will be described hereinafter which form the subject ofthe claims of the invention. It should be appreciated by those skilledin the art that the conception and the specific embodiment disclosedmight be readily utilized as a basis for modifying or redesigning thecomposition and method for carrying out the same purposes as theinvention. It should be realized by those skilled in the art that suchmodified or redesigned compositions and methods do not depart from thespirit and scope of the invention as set forth in the appended claims

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, and 1D shows the nucleotide and amino acid sequence ofa colanic acid-degrading enzyme according to one embodiment.

FIG. 2 shows a gel where several different DNA plasmid samples weretested using this gel electrophoretic method for polysaccharidevisualization and quantification.

FIGS. 3-8 show the polypeptide sequences of SEQ ID NOS: 1-6.

FIGS. 9-10 show the polynucleotide sequences of SEQ ID NO: 7 and SEQ IDNO: 8.

FIG. 11 shows the polypeptide sequences of SEQ ID NOS: 9-17.

FIGS. 12-14 show tables of results from post-iv injections of 120 micewith high doses of DNA-BIV liposomal complexes, purified according tothe processes described herein.

ABBREVIATIONS AND DEFINITIONS

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g. aCAE-related protein). For example an analog of a colonic acid degradingenzyme (CAE) protein can be specifically bound by an antibody or T cellthat specifically binds to CAE.

The term “antibody” is used in the broadest sense. Therefore an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-CAEantibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen-binding domain and/or one or morecomplementarity determining regions of these antibodies. An “antibodyfragment” is defined as at least a portion of the variable region of theimmunoglobulin molecule that binds to its target, i.e., theantigen-binding region. In one embodiment it specifically covers singleanti-CAE antibodies and clones thereof (including agonist, antagonistand neutralizing antibodies) and anti-CAE antibody compositions withpolyepitopic specificity.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used inthe context of nucleic acids, are meant to refer to conventionalhybridization conditions, preferably such as hybridization in 50%formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures forhybridization are above 37° C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55° C.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment. For example, a nucleic acid or polynucleotide is said to be“isolated” when it is substantially separated from contaminantpolynucleotides that correspond or are complementary to genes other thanthe target genes or that encode polypeptides other than the target geneproduct or fragments thereof. A skilled artisan can readily employnucleic acid isolation procedures to obtain an isolated polynucleotide.A protein is said to be “isolated,” for example, when physical,mechanical or chemical methods are employed to remove the targetproteins or polypeptides from cellular constituents that are normallyassociated with the protein. A skilled artisan can readily employstandard purification methods to obtain an isolated protein.Alternatively, an isolated protein can be prepared by chemical means.

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The term “monoclonal antibody” refers to a collection of antibodiesencoded by the same nucleic acid molecule which are optionally producedby a single hybridoma or other cell line, or by a transgenic mammal suchthat each monoclonal antibody will typically recognize the same epitopeon the antigen. The term “monoclonal” is not limited to any particularmethod for making the antibody, nor is the term limited to antibodiesproduced in a particular species, e.g., mouse, rat, etc. The term“polyclonal antibody” refers to a heterogeneous mixture of antibodiesthat recognize and bind to different epitopes on the same antigen.Polyclonal antibodies may be obtained, for example, from crude serumpreparations or may be purified using, for example, antigen affinitychromatography, or Protein A/Protein G affinity chromatography.

The terms “percent (%) amino acid sequence identity” with respect to thepolypeptide sequences identified herein, is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in a specific polypeptide sequence, after aligningthe sequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST™, BLAST-2™, ALIGN™,ALIGN-2™, or MEGALIGN™ (DNASTAR™) software. Persons of ordinary skill inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared. For instance, percent aminoacid sequence identity may be determined using the sequence comparisonprogram NCBI-BLAST2™ (Alschul et al., Nucleic Acids Res. 25:3389-3402(1997)). The NCBI BLAST2™ sequence comparison program may be obtainedfrom the National Institute of Health, Bethesda, Md. NCBI-BLAST2™ usesseveral search parameters, wherein all of those search parameters areset to default values including, for example, unmask=yes, strand=all,expected occurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

The terms “percent (%) nucleic acid sequence identity” with respect tothe polypeptide-encoding nucleic acid sequences identified herein, isdefined as the percentage of nucleotides in a candidate sequence thatare identical with the nucleotides in a specific polypeptide nucleicacid sequence of interest, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent nucleic acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST™, BLAST-2™, ALIGN™, ALIGN-2™, or MEGALIGN™ (DNASTAR™) software.Persons of ordinary skill in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared. For instance, percent nucleic acid sequence identity may alsobe determined using the sequence comparison program NCBI-BLAST2™(Alschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBIBLAST2™ sequence comparison program may be obtained from the NationalInstitute of Health, Bethesda, Md. NCBI-BLAST2™ uses several searchparameters, wherein all of those search parameters are set to defaultvalues including, for example, unmask=yes, strand=all, expectedoccurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide.”A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T) can also be uracil (U); this definition pertainsto the differences between the chemical structures of DNA and RNA, inparticular the observation that one of the four major bases in RNA isuracil (U) instead of thymidine (T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”.

A “recombinant” polynucleotide (e.g., DNA or RNA molecule) or“recombinant” polypeptide is a polynucleotide or polypeptide that hasbeen subjected to molecular manipulation in vitro.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C. “Moderately stringent conditions” are described by, but not limitedto, those in Sambrook et al., Molecular Cloning: A Laboratory Manual,New York: Cold Spring Harbor Press, 1989, and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent than those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the CAE-protein shown in FIG. 1 or FIG. 2). Ananalog is an example of a variant protein. Splice isoforms and singlenucleotides polymorphisms (SNPs) are further examples of variants.

The polypeptides having colanic acid-degrading (CAE) activity of theinvention include those specifically identified herein, as well asallelic variants, conservative substitution variants, analogs andhomologs that can be isolated/generated and characterized without undueexperimentation following the methods outlined herein or readilyavailable in the art. Fusion proteins that combine parts of differentCAE proteins or fragments thereof, as well as fusion proteins of a CAEprotein and a heterologous polypeptide are also included. Such CAEproteins are collectively referred to as the CAE-related proteins, theproteins of the invention, or CAE. The term “CAE-related protein” refersto a polypeptide fragment or a CAE protein sequence of at least 10, 15,25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,or more than 700 amino acids.

DETAILED DESCRIPTION

The present disclosure generally relates to “superclean” plasmid DNApreparations, processes for their preparation, and enzymes useful inthese processes. Polysaccharides, particularly colanic acid, have beenfound to contaminate purportedly “clean” preparations of plasmid DNAthat induce toxic effects in humans and mammals when used, e.g., forgene therapy. The components that are believed to induce these toxiceffects have not been identified in the literature. Other applicationsthat can be adversely affected by the presence of polysaccharidecontaminants in DNA preparations include clinical diagnostics,forensics, and other biotechnology methodologies, such as chips andmicroarrays including nucleic acids thereon, and in molecular studies(e.g., building chromosomes, analyses of transcriptional start sites,X-ray crystallography, and DNA structural studies, among others). Anurgent need exists to remove such contaminant molecules from DNApreparations.

Among other things, therefore, the present invention provides isolated,purified, and recombinant polypeptides having colanic acid-degradingactivity, and processes involving their use. The invention also providesisolated polynucleotides encoding such polypeptides. The polypeptidesdescribed herein have a range of uses, and enable processes fordigesting colanic acid in a biological material, and processes forremoving endotoxins from compositions including biologicalmacromolecules. Significantly, the polypeptides also enable thepreparation of plasmid DNA preparations, preferably gram negativebacterial plasmid DNA, comprising less than about 0.1 mg of colanic acidper mg of plasmid DNA, and more preferably less than about 0.05 mg ofcolanic acid per mg of plasmid DNA. In certain preferred embodiments, nodetectable colanic acid can be found in the plasmid compositionsprepared by the processes described herein. The plasmid compositions mayalso have very low levels, or undetectable levels, of otherpolysaccharide contaminants, such as uronic acid and fucose.

The disclosure relates, in part, to the discovery that the polypeptidecompounds of the invention are capable of digesting colanic acid (alsoknown as M-antigen), an exopolysaccharide produced by a range ofenterobacteria, including the majority of Escherichia coli strains.Depending on the bacteria, for example, colanic acid may be comprised offucose, glucose, galactose, and glucuronic acid, together with acetateand pyruvate, in various ratios (see, e.g., Sutherland, Biochem. J. 115,935-945 (1969). As noted above, endotoxins and polysaccharides, and inparticular colanic acid, have been found to contaminate preparations ofnucleic acids, e.g., for therapeutic uses, and it is difficult toseparate endotoxins and polysaccharides such as colanic acid fromnucleic acids using current standard purification procedures.

Accordingly, the polypeptides described herein may be generally used inthe digestion of colanic acid in a material. In general, any materialincluding colanic acid may be treated with the polypeptides describedherein; typically, the material is a biological material. The biologicalmaterial may be derived from, or a part of, microbes, tissues fromhumans and animals, and environmental samples such as archaeologicalremains, compost or other decomposing matter, peat bogs, plant matter,sediment, sludge, soil, and wastewater, e.g., that are terrestrial orsubterranean in origin. In certain embodiments, the biological materialis a biological slime. In accordance with these embodiments, forexample, the colanic acid may be present in the cellular membrane of theintact bacteria. In other embodiments, the biological material may be acrude bacterial lysate, a partially purified bacterial lysate, or anaqueous solution comprising extracted bacterial nucleic acid. In anotherembodiment, the biological material may be a biofilm. In anotherembodiment, the biological material may be pulp or pulp derivative(e.g., such as those mechanically or chemically prepared from wood orfiber sources). As noted above, in accordance with other aspects of theinvention, the polypeptides described herein may be used in processesfor the removal of endotoxins from aqueous compositions comprisingbacterial macromolecules (e.g., plasmid DNA). In certain preferredaspects, the polypeptides are used in processes for the purification ofplasmid DNA, typically gram negative bacterial plasmid DNA.

Plasmid DNA Compositions

One key aspect of the present disclosure is highly purified plasmidcompositions and pharmaceutical grade plasmid DNA compositions. Thesecompositions, for example, may generally be produced by the colanic acidenzymatic digestion processes described herein, which may or may not becombined with conventional purification techniques, such as one or morecombinations of chromatography and filtration steps. Thus, the inventionencompasses, or in addition comprises, a process of producing andisolating highly purified plasmid compositions that are essentially freeof polysaccharides including colanic acid, fucose, and uronic acid, andother contaminants, and thus is pharmaceutical grade DNA. In addition tohaving very low, and preferably undetectable, levels of colanic acid andother polysaccharides, the plasmid DNA produced and isolated by theprocesses described herein includes very low levels of endotoxingenerally, including one or more of contaminating chromosomal DNA, RNA,protein, and endotoxins, and preferably contains mostly closed circularform plasmid DNA. The plasmid DNA produced according to the processesdescribed herein is of sufficient purity for use research andplasmid-based therapy.

The plasmid compositions of the present invention may include any typesof vectors with any sizes. For instance, the size range of plasmid DNAthat may be purified by the processes described herein may be fromapproximately 0.3 kbp (mini-circle or minimal transcription unit) toapproximately 50 kbp, typically 3 kbp to 20 kbp, or larger (e.g., 5 to100 kbp, or larger, such as phage-derived shuttle vectors, HACs, YACs,MACs, and episomes derived from EBV or other non-integrating viruses).In certain embodiments, the DNA includes a vector backbone ofapproximately 0.3 kbp, 0.5 kbp, 0.75 kbp, 1 kbp, 3 kbp, 5 kbp, 10 kbp,15 kbp, or 20 kbp, a therapeutic gene, and associated regulatorysequences. This may also apply to single stranded DNA (i.e., 0.3 kb to50 kb, etc., such as those derived from M13). Thus, for example, avector backbone may be capable of carrying inserts of approximately 1-50kbp, or larger (e.g., 3-20 kbp), or approximately 1-50 kb, or larger(e.g., 3-20 kb). The insert will generally depend on application inwhich the plasmid composition is to be used. For gene therapy orvaccine-based applications, for example, the insert may include DNA fromany organism, but will typically be of mammalian origin, and mayinclude, in addition to a gene encoding a therapeutic protein,regulatory sequences such as promoters, poly adenylation sequences,enhancers, locus control regions, etc. The gene encoding a therapeuticprotein may be of genomic origin, and therefore contain exons andintrons as reflected in its genomic organization, or it may be derivedfrom complementary DNA. Such vectors may include for example vectorbackbone replicatable with high copy number replication, having apolylinker for insertion of a therapeutic gene, a gene encoding aselectable marker, e.g., the tetracycline or kanamycin resistance gene,and is physically small and stable. The vector backbone of the plasmidadvantageously permits inserts of fragments of mammalian, othereukaryotic, prokaryotic or viral DNA, and the resulting plasmid may bepurified as described herein and used in vivo or ex vivo plasmid-basedtherapy, or other use. The plasmid compositions can also comprise otherpharmaceutically acceptable components, buffers, stabilizers, orcompounds for improving gene transfer and particularly plasmid DNAtransfer into a cell or organism.

In general, “superclean” plasmid DNA compositions are provided herein.Typically, the plasmid DNA composition is a gram negative bacterialplasmid DNA composition. As described herein, an efficientenzymatically-based process has been developed that allows for theremoval of colanic acid contamination from a variety of bacterialmaterials, such as plasmid DNA samples. In various embodiments, thefirst step in the process may involve detection of colanic acid as asource of contamination (e.g., plasmid DNA contamination). This may bedirectly or indirectly accomplished, for example, by assaying for thepresence of fucose in the sample (described below), since fucose isknown to make up about 22% of colanic acid.

In one embodiment, the plasmid DNA composition is a gram negativebacterial plasmid DNA composition comprising gram negative bacterialplasmid DNA and less than about 0.1 mg of colanic acid per mg of gramnegative bacterial plasmid DNA. More preferably in this embodiment, thecomposition comprises less than about 0.05 mg of colanic acid per mg ofgram negative bacterial plasmid DNA. In one particularly preferredembodiment, the gram negative bacterial plasmid DNA compositioncomprises no detectable colanic acid.

As noted elsewhere herein, the plasmid DNA compositions of the inventionalso preferably include low or undetectable levels or otherpolysaccharide contaminants, such as uronic acid or fucose. Thus, in oneembodiment, the gram negative bacterial plasmid DNA compositioncomprises less than 0.1 mg of uronic acid per mg of gram negativebacterial plasmid DNA. More preferably in this embodiment, the gramnegative bacterial plasmid DNA composition comprises less than 0.05 mgof uronic acid per mg of gram negative bacterial plasmid DNA.Preferably, no detectable uronic acid can be found in the plasmid DNAcomposition.

In these and other embodiments, the gram negative bacterial plasmid DNAcomposition preferably comprises less than 0.1 mg of fucose per mg ofgram negative bacterial plasmid DNA. More preferably in this embodiment,the gram negative bacterial plasmid DNA composition comprises less than0.05 mg of fucose per mg of gram negative bacterial plasmid DNA.Preferably, no detectable fucose can be found in the plasmid DNAcomposition.

In combination, the gram negative bacterial plasmid DNA composition maycomprise less than about 0.1 mg of colanic acid per mg of gram negativebacterial plasmid DNA, and less than about 0.1 mg of uronic acid per mgof gram negative bacterial plasmid DNA. For instance, the gram negativebacterial plasmid DNA composition may comprise 0.05 mg of colanic acidper mg of gram negative bacterial plasmid DNA, and less than about 0.05mg of uronic acid per mg of gram negative bacterial plasmid DNA.Preferably, no detectable colanic acid and no detectable uronic acid ispresent in the gram negative bacterial plasmid DNA composition.

By way of another combination, the gram negative bacterial plasmid DNAcomposition may comprise less than about 0.1 mg of colanic acid per mgof gram negative bacterial plasmid DNA, and less than about 0.1 mg offucose per mg of gram negative bacterial plasmid DNA. For instance, thegram negative bacterial plasmid DNA composition may comprise 0.05 mg ofcolanic acid per mg of gram negative bacterial plasmid DNA, and lessthan about 0.05 mg of fucose per mg of gram negative bacterial plasmidDNA. Preferably, no detectable colanic acid and no detectable fucose ispresent in the gram negative bacterial plasmid DNA composition.

In yet another combination, the gram negative bacterial plasmid DNAcomposition may comprise less than about 0.1 mg of colanic acid per mgof gram negative bacterial plasmid DNA, less than about 0.1 mg of uronicacid per mg of gram negative bacterial plasmid DNA, and less than about0.1 mg of fucose per mg of gram negative bacterial plasmid DNA. Forinstance, the gram negative bacterial plasmid DNA composition maycomprise 0.05 mg of colanic acid per mg of gram negative bacterialplasmid DNA, less than about 0.05 mg of uronic acid per mg of gramnegative bacterial plasmid DNA, and less than 0.05 mg of fucose per mgof gram negative bacterial plasmid DNA. Preferably, no detectablecolanic acid, no detectable uronic acid, and no detectable fucose ispresent in the gram negative bacterial plasmid DNA composition.

In addition to the reduced levels of colanic acid, uronic acid, and/orfucose discussed above, the plasmid compositions described herein mayalso include, for example, less than 0.01 mg chromosomal or genomic DNA,RNA, protein, and/or endotoxin contaminants per mg of gram negativebacterial plasmid DNA; more preferably, the composition includes lessthan 0.001 mg, less than 0.0001 mg, or less than 0.00001 mg chromosomalor genomic DNA, RNA, protein, and/or endotoxin contaminants per mg ofgram negative bacterial plasmid DNA. In one embodiment, for example, theplasmid compositions may comprise less than 0.1 mg (more preferably,less than 0.05 mg; still more preferably, no detectable amount) ofcolanic acid per mg of gram negative bacterial plasmid DNA, and lessthan 0.01 mg (more preferably, less than 0.001 mg; still morepreferably, 0.0001 mg) host cell chromosomal DNA or genomic DNAcontaminants per mg of gram negative bacterial plasmid DNA. The plasmidcomposition may also comprise less than 0.1 mg (more preferably, lessthan 0.05 mg; still more preferably, no detectable amount) of colanicacid per mg of gram negative bacterial plasmid DNA composition, and lessthan 0.01 mg (more preferably, less than 0.001 mg; still morepreferably, 0.0001 mg) host cell protein contaminants per mg of gramnegative bacterial plasmid DNA.

Assays for detecting levels of colanic acid, uronic acid, fucose, andother polysaccharides are generally known in the art (or are describedherein); methods of detecting chromosomal or genomic DNA, RNA, protein,and/or endotoxin that may be present in the plasmid compositions arealso generally known in the art.

In one embodiment, for example, the plasmid composition of the presentinvention may include, less than 0.1 mg, preferably less than 0.05 mg ofcolanic acid per mg of gram negative plasmid DNA (e.g., 0.04, 0.03,0.02, or 0.01 mg), and more preferably no detectable colanic acid, asmeasured by a bicinchoninic acid (BCA) assay. Suitable BCA assays aredescribed, for example, in Meeuwsen et al., Biosci. Bioeng. 89, 107-109(2000); and Verhoef et al., Carbohyd. Res. 340(11), 1780-1788 (2005). Anexemplary BCA assay for measuring colanic acid levels is found inExample 16.

In another embodiment, the plasmid composition may include colanic acidat the levels recited in the previous paragraph, and further compriseless than about 0.1 mg, preferably less than about 0.05 mg of uronicacid per mg of gram negative plasmid DNA (e.g., 0.04, 0.03, 0.02, or0.01 mg), and more preferably no detectable uronic acid, as measured bya uronic acid assay. In general, the uronic acid content of a plasmidDNA sample is measured using standard curves generated with heparinsulfate or glucuronic acid as standards. For instance, heparin sulfateresembles the polysaccharide contaminants from E. coli, because uronicacid comprises about 25% of the total weight of heparin sulfate. Heparinsulfate consists of 50% sugars by weight. Half of these sugars areglucosamine and the other half of the sugars are iduronic acid andglucuronic acid; the rest of the heparin sulfate is contributed bymodifications of the sugars including sulfates and acetylamides.Alternatively, glucuronic acid can be used to create a standard curvefor the direct measurement of uronic acid. The standard solution isplaced is a glass test tube with a borate/sulfuric acid solution (e.g.,0.025 M sodium tetraborate 10-hydrate dissolved in sulfuric acid havinga specific gravity of 1.84) and mixed. A solution of carbazole inabsolute ethanol is added to the mixture and the entire mixture isvortexed and immersed in boiling water. The tubes are allowed to cooland the absorbance of the solution at 530 nm is read in aspectrophotometer. The absorbance values obtained for the standards areplotted against the concentration of the standards. The uronic acidcontent of plasmid DNA samples can be extrapolated from its absorbancevalue at 530 nm when the DNA sample has undergone the same reaction. Thepolysaccharide content of the plasmid DNA sample can then beextrapolated by multiplying the amount of uronic acid by a numberranging from 3.3 to 9.1 (depending on the prevalence of colanic acid,ECA and the O- and K-antigens in the sample). An exemplary uronic acidassay for measuring uronic acid levels is found in Example 16.

In another embodiment, the plasmid composition may include colanic acidand/or uronic acid at the levels recited in the previous paragraphs, andfurther comprise less than about 0.1 mg, preferably less than about 0.05mg of fucose per mg of gram negative plasmid DNA (e.g., 0.04, 0.03,0.02, or 0.01 mg), and more preferably no detectable fucose, as measuredby a fucose assay. The basic procedures for assay of fucose content insamples can be found in Morris, Anal. Biochem. 121, 129-134 (1982).Detailed descriptions of the solution preparation and storage conditionsfor solutions and samples for this assay have also been published(Passonneau, J. V. and O. H. Lowry. 1974. In: Methods of EnzymaticAnalysis, U. H. Bergmeyer (ed.), 2nd edition, Academic Press: New York,volume 4, pp. 2059-2072). Using this method, the fucose levels ofplasmid DNA samples were determined and the concentration of colanicacid levels calculated. As described elsewhere herein, colanic acid wasfound to be the primary contaminant in plasmid DNA from a variety ofsources, even GMP grade plasmid DNA. An exemplary fucose assay formeasuring fucose levels is found in Example 16.

In these and other embodiments, the gram negative plasmid compositionpreferably comprises no visually detectable polysaccharides whencombined with a polysaccharide-selective labeling agent. In general,polysaccharide visualization assays involve labeling polysaccharideswith a fluorescent agent and detecting their presence, e.g., on aelectrophoretic medium. Preferably, no polysaccharides are detectableusing such agents in combination with the plasmid compositions describedherein. In one embodiment, the polysaccharide-selective labeling agentis (4,6-dichlorotriazinyl)aminofluorescein (DTAF). Otherpolysaccharide-selective labeling agents are known or will be evident tothose skilled in the art. An exemplary assay utilizing apolysaccharide-selective labeling agent is found in Example 16.

In addition, viscosity of plasmid DNA compositions are reduced as aresult of the colanic acid degradation processes described herein.Accordingly, the presence of colanic acid may be detected by comparingthe viscosity of a treated plasmid DNA composition with the viscosity ofan untreated plasmid DNA sample. An exemplary assay utilizing viscositylevels of plasmid DNA is described in Example 16.

In general, as the levels of colanic acid (and/or the levels of uronicacid, fucose, or other polysaccharide or other contaminant) present inthe plasmid composition are reduced, the LD₅₀ (the dose lethal to 50% ofthe test population) of the plasmid composition is increased. Forinstance, by reducing the level of colanic acid to less than 0.1 mg permg of gram negative bacterial plasmid DNA, in one embodiment thecorresponding LD₅₀ of the plasmid composition is increased by at least25%; more preferably in this embodiment, by at least 50%. By furtherreductions in the levels of colanic acid (and other polysaccharides),and optionally similar reductions in the levels of other contaminantssuch as chromosomal or genomic DNA, RNA, protein, and/of endotoxin, thecorresponding LD₅₀ of the plasmid composition may be increased by atleast 50%, by at least 75%, or by at least 100%. By way of anotherexample, by reducing the level of colanic acid to less than 0.05 mg permg of gram negative bacterial plasmid DNA and the level of chromosomalor genomic DNA, RNA, protein, and/or endotoxin contaminants to less than0.01 mg per mg of gram negative bacterial plasmid DNA, the correspondingLD₅₀ of the plasmid composition may be increased by at least 50%; morepreferably in this embodiment, at least 100%.

In general, the plasmid compositions of the present invention may beutilized in a wide range of applications, including those in fields ofbioterrorism (agent detection and analysis), environmental science(e.g., agriculture, horticulture, and forestry), food science,forensics, molecular biology, health and medicine (e.g., gene therapy,diagnostics, recombinant protein expression), and space science, to namea few. The highly pure plasmid compositions described herein may beemployed in vivo or ex vivo, for example, in gene therapy andvaccine-based applications (i.e., the plasmid compositions may beadministered to mammals, including humans). Additionally oralternatively, the plasmid compositions may be used in conventionaldiagnostics and forensics techniques, for example, to improve thestability, specificity, reproducibility, and/or sensitivity of suchmethodologies. This may include, for example, the analysis, detection,or examination of samples from the environment, e.g. from public watersupplies, samples from foodstuffs, and from other biological or clinicalsamples, such as blood, saliva, sputum, semen, buccal smears, urine orfecal waste, cell and tissue biopsies and micro dissections, amnioticfluid, or tissue homogenates of plants, animals, or human patients, andthe like. Other examples of uses for the plasmid compositions describedherein include genotyping microorganisms, DNA fingerprinting of plantsand animals, detecting pathogens and beneficial microorganisms in soils,water, plants and animals, forensic identification of biological samplesand environmental samples contaminated with different biologicalentities, and molecular studies such as, for instance, buildingchromosomes, analyses of transcriptional information, X-raycrystallography, and DNA structural studies. The plasmid compositionsmay also be used in conjunction or in combination with solid substratechip formats that detect, among other things, genes, mutations or mRNAexpression levels such as nucleic acid microarrays and moleculardetection chips employing, for example, fluorescence, radioactivity,optical interferometry, Raman spectrometry, semi-conductor, or otherelectronics (see, e.g., U.S. Pat. No. 7,098,286; U.S. Pat. No.6,924,094; and U.S. Pat. No. 6,824,866 (each of which is herebyincorporated by reference herein)).

Processes

As described elsewhere herein, the polypeptides of the present inventioncan be utilized in a wide range of processes, particularly those whichinvolve, require, or otherwise benefit from digestion or degradation ofcolanic acid, typically in a biological material, such as a bacterialsample, or compositions (such as aqueous compositions) comprisingbacterial macromolecules. In general, biological material includingundesirable colanic acid may be treated with the polypeptides describedherein, including biofilms (i.e., structured communities ofmicroorganisms encapsulated within self-developed polymeric matrices,either adherent to a living or inert surface, or on its own), bacteriallysates, plasmid DNA, and the like.

The processes described herein generally involve the digestion ofcolanic acid in a biological material, or otherwise in a compositioncomprising a biological material. In general, the processes employ thepolypeptides described herein to digest or degrade colanic acid that maybe present in the material. One embodiment of the processes describedherein involves, for example, the digestion of colanic acid from abiological material. Alternatively, the processes may involve thedigestion of colanic acid in an aqueous composition containing bacterialmacromolecules. By way of another alternative, the processes may involvetreating an aqueous composition containing plasmid DNA with apolypeptide to digest colanic acid.

A polypeptide is used to digest colanic acid present in the sample;thus, the polynucleotide has colanic acid-degrading activity, or isotherwise a colanic acid-degrading enzyme. In one particular embodiment,the process involves digesting in a biological material and the processcomprises contacting the biological material with a polypeptide capableof digesting colanic acid. The biological material may be, for example,a crude bacterial lysate, a partially purified bacterial lysate, and anaqueous solution containing extracted bacterial nucleic acid (such asgram negative plasmid DNA). Alternatively, the biological material maybe a bacterial slime. By way of another alternative, the biologicalmaterial may be a biofilm comprising gram negative bacteria. In anotheralternative embodiment, the bacterial material may be present in a pulp(e.g., wood or fiber pulp) composition, solution, or mixture. In anotherembodiment, the process involves the removal of endotoxin from anaqueous composition containing bacterial macromolecules and the processcomprises digesting colanic acid in the aqueous composition andthereafter combining the aqueous composition with a chromatographicmaterial to separate endotoxin from the bacterial macromolecule. In onepreferred embodiment, the process involves purification of plasmid DNA,such as gram negative bacterial plasmid DNA, and the process comprisestreating an aqueous composition containing plasmid DNA with apolypeptide to digest colanic acid and separating the plasmid DNA fromthe treated aqueous composition using, for example, conventionalchromatography techniques. The polypeptide may also be employed in arange of industrial processes described below.

In a particular aspect, a process for removal of contaminatingpolysaccharides from plasmid DNA samples has been developed that allowsfor the removal of polysaccharides, including those other than LPS, fromplasmid DNA samples. In addition, RNA and LPS are also removed from theplasmid DNA samples. Therefore, the method of the present inventionresults in purified plasmid DNA that contains extremely low, and in manycases undetectable, levels of polysaccharides as described below. Unlikeprevious methods, which were unable to identify the levels ofcontaminating polysaccharides, the certain steps may be performed toquantify the polysaccharide levels in DNA samples, allowing theinvestigator to assure the removal of polysaccharides from the DNAsample.

In general, any of the polypeptides described herein may be employed.For example, the polypeptide may comprise an amino acid sequence havingat least 90% homology to SEQ ID NO: 1, or the polypeptide may comprisean amino acid sequence having at least 90% homology to SEQ ID NO: 2. Inone particular embodiment, the polypeptide has the amino acid sequenceof SEQ ID NO: 1. In another particular embodiment, the polypeptide hasthe amino acid sequence of SEQ ID NO: 2.

The starting material for certain of the processes described herein is amass of bacterial material, or an aqueous composition comprising suchbiological material, such as bacterial cells or other biological matterprepared by, e.g., fermentation or cell culture, isolated from theenvironment, or derived from tissues or other organisms (e.g., fungi,bacteria, etc.). In one embodiment, the biological material comprisesbacterial cells derived from enterobacteria, such as E. coli. In anotherembodiment, the biological material is a bacterial slime; according tothis embodiment, for example, colanic acid is present in the cellularmembrane of the bacteria. In a preferred embodiment, the biologicalmaterial is a gram negative bacterial plasmid DNA material.

A variety of cell types can be used as feed for the processes describedherein, such as bacteria (e.g., gram (−), gram (+), and Archaea), yeast,and other prokaryotic and eukaryotic cells, including mammalian cellsand recombinant cells. Among these and other cell types, bacterialcells, and in particular gram positive (+) and gram negative (−)bacterial cells, such as E. coli, Salmonella, or Bacillus, arepreferred, with gram negative (−) bacterial cells being most preferred.In a particular embodiment, the bacteria is a gram negative (−)bacteria; more preferably in this embodiment, the bacteria is E. coli. Awide selection of well-established E. coli host strains are usefulaccording to the processes described herein, and are available fromStratagene (La Jolla, Calif.), Qiagen (Valencia, Calif.), New EnglandBioLabs (Ipswich, Mass.), and Promega (Madison, Wis.), among othercommercial sources.

Typically, the biological material is a bacterial lysate, or aderivative thereof. Thus, bacterial starting material (e.g., bacterialcells, etc.) must be lysed or disrupted to form the lysate. In general,the bacterial lysis step involves any conventional method for breakingopen bacterial cells, thus liberating nucleic acids and other cellcomponents therefrom. The lysis procedure may involve the use ofmechanical methods, lysing agents or solutions, or combinations thereof.

For biological material derived from fermentation or cell culture, thecells are disrupted by chemical or mechanical techniques as describedbelow, forming a crude lysate. For example, where bacterial cultures areemployed, the bacterial cells are lysed to form a crude bacteriallysate. In doing so, the cellular components, including DNA, RNA,proteins, colanic acid, and other polysaccharides, are released from thecells. In certain embodiments, the lysate may undergo pre-treatmentsteps, such as purification steps to remove cell contaminants andendotoxins, thus forming a partially purified (e.g., bacterial) lysate.

Where a lysing agent is employed, the lysing agent is used to break downcell membranes, thus releasing DNA, RNA and proteins from the cells. Onepreferred lysing agent comprises an alkaline solution. A variety ofbases may be employed in conventional alkaline lysis procedures,including, for example, hydroxide salts, such as potassium hydroxide(KOH), lithium hydroxide (LiOH), or sodium hydroxide (NaOH). Typically,the base is sodium hydroxide. Often, detergents are employed in lysingsolutions, either alone or in combination with the alkaline solution. Ingeneral, and depending on the application, the detergent may be ancationic, anionic, non-ionic, or zwitterionic detergent, or acombination thereof. One exemplary anionic detergent is sodium dodecylsulfate (SDS). One exemplary zwitterionic or non-ionic detergent isTween 20.

Mechanical methods for lysing bacterial cells, for use either alone orin combination with lysis solutions and agents, include agitation,sonication, centrifugation, freeze/thawing, French cell press, and thelike.

Alkaline lysis and mechanical techniques for lysing bacterial cells torelease and extract proteins and nucleic acids are generally well known,and are described, for example, in Sambrook et al., supra.

In some embodiments, it may be desirable to form a cleared lysatepreparation, in which the chromosomal DNA, proteins, and membraneportions of the host cells have been at least partially removed, such asby chemical treatment or centrifugation of the lysate, thereby leaving asolution containing plasmid DNA. RNAse can optionally be added atvarious points in the procedure to create a cleared lysate that issubstantially free of RNA. As noted elsewhere herein, initial removal ofmany cellular and nucleic acid contaminants can improve colanic aciddigestion and/or further purification of the plasmid DNA usingconventional chromatographic techniques. Methods of creating clearedlysates are well-known in the art. For example, a cleared lysate can beproduced by treating the host cells with sodium hydroxide or itsequivalent (0.2N) and sodium dodecyl sulfate (SDS) (1%), centrifuging,and discarding the supernatant. This method of creating a cleared lysateis generally described, for example, in Burnboim et al., Nucl. AcidsRes., 7, 1513 (1979); and Horowicz et al., Nucl. Acids Res., 9, 2989(1981).

For many uses, e.g., therapeutic uses such as in gene therapy or theformation of vaccines, it may be desirable to further purify the nucleicacid obtained from the bacterial or other lysate, either before or afterthe sample is contacted with the colanic acid-degrading polypeptide.

After forming the bacterial lysate as described above, it is generallypreferably to subject the crude lysate to separation techniques toeliminate at least part of the other nucleic acid, protein, and cellcontaminants that are present, before attempting to digest colanic acidwith the polypeptide. Thus, in one embodiment, for example, the materialor composition comprising the biological matter is combined with an ionexchange chromatographic material prior to treatment with thepolypeptide. Typically, the chromatographic material is a anion exchangechromatographic resin. In a preferred embodiment, the anion exchangechromatographic resin comprises diethylaminoethyl cellulose (DEAE). Ingeneral, conventional DNA, including plasmid DNA, cleaning techniquescan be employed.

The biological material may also be combined with chromatographicmaterial following treatment with the polypeptide. This generallyinvolves affinity chromatography and/or hydrophobic interactionchromatography. In a particular embodiment, the biological material oraqueous composition is combined with an anion exchange chromatographyresin, treated with the polypeptide, combined with an affinitychromatography resin, combined with a hydrophobic interactionchromatography resin, and subjected to filtration, in that order.

Once the biological material is lysed or otherwise prepared, thematerial is treated with the polypeptide described herein. Typically,treatment involves contacting or mixing the polypeptide with thebiological material such that the polypeptide will have access to thecolanic acid substrate present in the material or composition.Preferably, the biological material and the polypeptide are incubated toallow sufficient interaction between the enzyme and the colanic acidsubstrate. Typically, the duration of the incubation may be from 1 to 6hours, 6 to 12 hours, 12 to 24 hours, or longer, depending on the sizeof the sample to be digested, the amount of polypeptide employed, andenvironmental factors, such as temperature, atmosphere, etc. Theincubation is generally carried out at a temperature of between 0° C.and 100° C., more preferably between 25° C. and 75° C. (e.g., between30° C. and 50° C.). In some embodiments, it may be desirable to vary thetemperature during the incubation process, for example, starting theincubation at a cooler temperature, and then raising the temperature forthe remainder of the incubation cycle, or vice versa.

As noted above, it will generally be desirable to purify the biologicalmaterial or aqueous composition containing biological material, eitherbefore or after treatment with the polypeptides of the invention. Ingeneral, this may involve subjecting the material or composition to oneor more chromatography methods or to filtration. In one embodiment, acombination of chromatographic separations are employed. Thus, forexample, at various times during the process, the biological material orcomposition may be combined with a chromatographic material. Suitablechromatographic materials include, for example, ion exchangechromatography resins (such as anion exchange chromatography resins andcation exchange chromatography resins), hydrophobic interactionchromatography resins, and affinity chromatography resins, among a rangeof others. In one embodiment, the chromatographic material is selectedfrom the group consisting of an anion exchange chromatography resin, acation exchange chromatography resin, a hydrophobic interactionchromatography resin, and an affinity chromatography resin.

As noted above, the biological material or composition may be combinedwith the chromatographic material(s) prior to, or after, treatment withthe polypeptide, or both before and after treatment with thepolypeptide, with single or multiple chromatographic separations beingemployed. For example, the sample can be combined with a chromatographymaterial, treated with the polypeptide as described herein, andsubjected to a second (or third, fourth, fifth, etc.) chromatographystep. In addition, the sample can be subjected to conventionalfiltration techniques to further purify or remove contaminants from thesample. The filtration steps may occur relatively early in the process,e.g., prior to treatment with the enzyme, or later in the process, e.g.,as final filtration steps prior to storage or use of the end product.

In another aspect, therefore, the processes of the invention comprisethe use of an polypeptide capable of degrading colanic acid present in abacterial lysate sample, which is preceded by or followed by at leastone additional chromatography technique. The additional chromatographystep may exist optionally or typically as one or more of the finalpurification steps or at least at the end or near the end of the sampleor plasmid purification scheme, or prior to the colanic acid digestionstep. In combination with the colanic acid degradation step, therefore,is preferably one or more of ion exchange chromatography, affinitychromatography (e.g., boronate affinity chromatography), hydrophobicinteraction chromatography, and filtration. Other techniques may includegel permeation or size exclusion chromatography, hydroxyapatite (type Iand II) chromatography, and reversed phase chromatography. In general,any available chromatography protocol involving nucleic acid separationcan be adapted for use. In addition, any one or more of the steps ortechniques can employ high performance chromatography techniques orsystems. Thus, the method of the invention comprises an colanic aciddigestion step with one or more step of ion exchange chromatography andfurther may include affinity chromatography, hydrophobic interactionchromatography or gel permeation chromatography, and/or filtration (suchas tangential flow filtration (TFF) or size exclusion filtration). Thestep of ion exchange chromatography, for example, may be both influidized bed ion exchange chromatography and axial and/or radial highresolution anion exchange chromatography. In one preferred embodiment,ion exchange chromatography is performed prior to the colanic aciddegradation step, in order to remove particles that may hinder theability of the enzyme to interact with the substrate.

Processes of the invention described herein, e.g., for purifying plasmidDNA, are scalable and thus amenable to scale-up to large-scalemanufacture.

In some embodiments of the invention, colanic acid degradation step maybe combined with additional purification steps to result in a highpurity product containing plasmid DNA. It may, for example, be combinedwith at least one of flocculate removal (such as lysate filtration,settling, or centrifugation), ion exchange chromatography (such ascation or anion exchange), and hydrophobic interaction chromatography.In one embodiment, the colanic acid degradation step is preceded by ionexchange chromatography. In these and other embodiments, the colanicacid degradation step is followed by hydrophobic interactionchromatography. In a preferred embodiment, bacterial lysis is followedby ion exchange chromatography, ion exchange chromatography is followedby affinity chromatography (e.g., using boronates or other vicinial- orcis-diol specific compounds), which is followed by hydrophobicinteraction chromatography. After or between one or more of these steps,the sample can be subjected to filtration, such as tangential flowfiltration. These steps allow for a truly scaleable plasmidmanufacturing process, which can produce large quantities of plasmid DNAwith high levels of purity. Host cell DNA and RNA, proteins, endotoxins,and colanic acid contaminants are preferably undetectable.

As noted above, the method of the present invention may also use furthersteps of size exclusion chromatography (SEC), reversed-phasechromatography, hydroxyapatite chromatography, and/or other availablechromatography techniques, methods, or systems in combination with thesteps described herein in accordance with the present application.

A flocculate removal step may also be employed to provide higher purityto the resulting plasmid DNA product. This step may be used to remove alarge portion of precipitated material (flocculate). One mechanism ofperforming flocculate removal is through a lysate filtration step, suchas through a 1 to 5 mm, and preferably a 3.5 mm grid filter, followed bya depth filtration as a polishing filtration step. Other methods ofperforming flocculate removal are through centrifugation or settling.Alternatively, the flocculate may be removed by ion exchangechromatography.

At various times in the endotoxin removal and/or plasmid DNApurification process, therefore, the sample may be subjected to one ormore of ion exchange chromatography (e.g., anion or cation exchangechromatography), affinity chromatography, hydrophobic interactionchromatography, and filtration (e.g., filtered through a 0.2 μm and/or0.45 μm filter), and, optionally, filtered or subjected tochromatography methods a second, third, or fourth time, or more.

Thus, for example, the sample can be subjected to a first chromatographymaterial, treated with the polypeptide, subjected to a secondchromatography material, and subjected to a third chromatographymaterial. The sample may also be filtered at various times in thesequence, such as after the first chromatographic separation, or afterthe third chromatographic separation. It will be understood thatsubjecting the sample to a chromatographic material also involveseluting the desired portion of the sample (such as that portioncontaining purified plasmid DNA) from the chromatographic material, anddiscarding undesired portions of the sample. Depending on the nature ofthe chromatographic material, the desired portions may be retainedwithin the chromatographic material and eluted in a separate step whilethe undesired materials flow through the material, or the undesiredmaterials may be retained in the chromatographic material while thedesired portions flow through the chromatographic material.

At a variety of places in the above protocol, analytical determinationof nucleic acid yield and purity are advantageously performed.Typically, such assays are performed before and after each purificationstep, as well as to each nucleic acid-containing fraction from, e.g.,preparative ion exchange chromatography or filtration. Representativemeans for performing these analytical determinations include HPLCanalysis of purity, spectrophotometric estimation of yield, silverstaining and SDS-PAGE for protein analysis, and agarose gelelectrophoresis and Southern blotting for DNA analysis. In certainembodiments, the processes described herein yields a purifiedconcentrate with a plasmid DNA concentration (including, for example,predominantly supercoiled or other plasmid DNA) of around 70%, 75%, 80%,85%, 90%, 95%, and preferably 99%, or greater.

Additionally, it is also generally desirable to analytically determinethe presence of polysaccharides such as colanic acid, uronic acid,and/or fucose in the intermediate or end products. Suitable assays forthe detection of colanic acid and other polysaccharides are describedelsewhere herein (e.g., in Example 16).

Particular chromatographic and filtration techniques are described infurther detail below.

Ion Exchange Chromatography

As noted above, ion exchange chromatography can be employed to purifythe biological sample prior to treatment with the polypeptide, orthereafter. This process generally separates nucleic acid, e.g., plasmidDNA, in the bacterial lysate or lysate derivative from contaminatingendotoxin, trace proteins, and residual cellular contaminants. Cation oranion exchange may be selected depending on the properties of thecontaminants and the pH of the solution. Anion exchange chromatography,for example, functions by binding negatively charged (or acidic)molecules to a support which is positively charged. The use ofion-exchange chromatography, then, allows molecules to be separatedbased upon their charge. Families of molecules (acidics, basics andneutrals) can be easily separated by this technique. Stepwise elutionschemes may be used, with many contaminants eluting in the earlyfractions and the plasmid DNA eluted in the later fractions. Ionexchange chromatography is a relatively common method for removingproteins and endotoxin from plasmid DNA preparations. The ion exchangechromatography or any one or more of the other chromatography steps ortechniques used can employ stationary phases, displacementchromatography methods, simulated moving bed technology, and/orcontinuous bed columns or systems.

The ion exchange columns that can be utilized in the processes of thepresent invention include both cationic and anionic ion exchangecolumns. In more preferred embodiments, the ion exchange chromatographymaterial is anion exchange chromatography resin. For example, the anionexchange chromatography material resin may comprise diethylaminoethylcellulose (DEAE), trimethylaminoethyl (TMAE), quaternary amino ethyl(QAE), or polyethyl imide (PEI) resins. In one embodiment, the anionexchange chromatography resin comprises DEAE. In another embodiment, theanion exchange chromatography resin comprises a quaternary ammoniumresin. For instance, a chromatography column may be packed with one ormore an anion exchange chromatography resins described herein. Theoptimal capacity of the column will be determined empirically based onthe resin used and the size of nucleic acid to be purified. For manyplasmid DNAs, preferred resins are those with no pore or with a largepore size, e.g., greater than 1000 Å, preferably around 3000 Å to 4000Å; with a medium bead size, e.g., about 20 to 500 μm diameter; that doesnot leach matrix components. Ideally, the resin is also washable, e.g.,with sodium hydroxide, to allow repeated use.

Relatively weak cationic and strong cationic ion exchange columns canalso be used. Relatively strong cationic exchange columns typically havea surface coated with a polyhydroxylated polymer and functionalized withsulfopropyl, a dextran matrix functionalized with a sulfopropyl group,or a surface coated with a polyhyroxylated polymer functionalized withsulfoethyl. Examples of strong cationic ion exchange columns using eachof these materials include, respectively, a POROS HS™, a POROS S™, and aSP SEPHADEX™ column, among others. Relatively weak cationic exchangecolumns typically have a dextran matrix functionalized by carboxymethylor an acrylic matrix functionalized by a carboxylic group. Examples ofweak cationic exchange columns using each of these materials include,respectively, CM SEPHADEX™ and BIO-REX 70™.

Relatively weak anionic and strong anionic ion exchange columns can alsobe used in the methods of the present invention. Weak anionic ionexchange columns typically have a surface coated with polyethyleneiminethat is capable of surface ionization up to a pH of about 9, astyrenedivinylbenzene copolymer containing sulfonic acid groups or adextran matrix functionalized by diethylaminoethyl. Examples of weakanionic exchange columns using each of these materials include,respectively, a POROS PI™ column, a DOWEX 50™ column and a DEAESEPHADEX™. Relatively strong anionic exchange columns typically have asurface coated with quaternized polyethyleneimine with a surfaceionization over a pH range of about 1 to about 14. An example of such astrong anionic ion exchange column is a POROS HQ™ column, or a SOURCE™column. The resins for the columns listed above can be obtained fromAmersham/Pharmacia (Piscataway, N.J.), PerSeptive Biosystems (FosterCity, Calif.), Toso Haas (Montgomeryville, Pa.), GE Healthcare(Piscataway, N.J.), and other suppliers.

Typically, the sample is combined with an ion exchange chromatographyresin that is present in a column. The column can be a 0.5 ml column, a1.5 ml column, a 10 ml column, a 20 ml column, a 30 ml column, a 50 mlcolumn, a 100 ml column, a 200 ml column, a 300 ml column, a 400 mlcolumn, a 500 ml column, a 600 ml column, a 700 ml column, an 800 mlcolumn, a 900 ml column, a 1000 ml (1 L) column a 2000 ml (2 L) a 10 Lcolumn, a 20 L column, a 30 L column, a 40 L column, a 50 L column, a 60L column, a 70 L column, an 80 L column a 90 L column, a 100 L column,or a column with a capacity greater than 100 L, as well as any othercolumn with a capacity between the volumes listed above.

Typically, the ion exchange chromatography material is equilibratedprior to use at a pH ranging from about 6.0 to about 7.2 and at a saltconcentration that can range from about 100 mM to 200 mM. Therefore, thecolumn can be equilibrated at a pH of about 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2 or any other pH in between thesepH values and at a salt concentration of about 100 mM, 125 mM, 150 mM,175 mM, 200 mM or any other concentration in between the saltconcentration values listed above. Commonly used salts that can beutilized to equilibrate the chromatography material include NaCl, KCl orany other salt that can be adjusted to match the ionic strength of KCl.

For the ion exchange chromatography, packing material and method ofpreparing such material as well as process for preparing, polymerizingand functionalizing anion and cation exchange chromatography and elutingand separating plasmid DNA therethrough are well known in the art.

In addition, a chelating agent for bivalent metal ion may be used suchas for example, ethylenediamine-tetraacetic acid (EDTA), for inhibitingthe degradation of plasmids due to DNA-degrading enzymes in the lysateof Escherichia coli. The concentration of chelating agent for bivalentmetal ion is preferably 0.1 to 100 mM.

When applying the protein of interest to an anion exchange matrix anysuitable matrix can be employed including, but not limited to,aminoethyl, diethylaminoethyl, quaternary aminomethyl, quaternaryaminoethyl, diethyl-(2-hydroxypropyl)aminoethyl, triethylaminomethyl,triethylaminopropyl and polyethyleneimine exchangers, to achievefiltration of the protein of interest. Examples of commerciallyavailable anionic exchangers include the cellulose ion exchangers suchas DE32™ and DE52™ (WHATMAN, Florham Park, N.J.), the dextran ionexchangers such as DEAE-SEPHADEX C-25™, QAE-SEPHADEX C-25™,DEAE-SEPHADEX C-50™ and QAE-SEPHADEX C-50™ (Pharmacia, Piscataway,N.J.), the agarose or cross-linked agarose such as DEAE BIO-GEL A™(BIO-RAD, Hercules, Calif.), DEAE-SEPHAROSE CL-6B™ and Q-SEPHAROSE FASTFLOW™ (Pharmacia), the synthetic organic polymers, such as MONO Q™(Pharmacia), DEAE-5-PW™ and HRLC MA7P™ (BIO-RAD) and the coated silicamatrices such as DEAE Si5500™ and TEAP Si100™. Desirably, the anionexchange matrix is equilibrated in very low salt concentrations andemployed at an alkaline pH (e.g., pH 8.0 to 11.5) to facilitate bindingof acid and mildly basic contaminants.

When applying the protein of interest to a cation exchange matrix, it iscontemplated that any matrix functionalized with carboxymethyl,sulfonate, sulfoethyl or sulfopropyl groups can be employed. Desirably,the cation exchange matrix is equilibrated and employed at an acidic pH(e.g., pH 3.0 to 6.5) to facilitate binding of basic and mildly acidiccontaminants. Examples of commercially available cationic exchangers arethe cellulose-based CM23™, CM32™ AND CM52™ (WHATMAN); the dextran basedCM-SEPHADEX C-25™, SP-SEPHADEX C-50™ (Pharmacia); the agarose orcross-linked agarose-based CM BIO-GEL A™ (BIO-RAD), CM-SEPHAROSE FASTFLOW™ and S-SEPHAROSE Fast FLOW™ (Pharmacia); the synthetic organicpolymer-based MONO S™ (Pharmacia), SP-5-PW™ and HRLC MA7C™ (BIO-RAD) andthe coated silica matrices such as CM Si300™ and SP Si100™.

The sample (e.g., the lysate or derivative thereof, including thenucleic acid) is typically loaded onto the column in a loading buffercomprising a salt concentration below the concentration at which thenucleic acid would elute from the column. Generally, the saltconcentration will be in certain embodiments from about 10 to 50 mS,depending on the resin used. In general, for weaker anion-exchangeresins, a lower conductivity solution will be used, whereas for strongeranion-exchange resins, a higher conductivity solution will be used. Thecolumn will then be washed with several column volumes of buffer toremove those substances that bind weakly to the resin. Fractions arethen eluted from the column using a shallow continuous saline gradientaccording to conventional methods, for example, using up to 1.5M NaCl ina Tris-HCl buffer. Sample fractions are collected from the column. Forintermediate scale preparations (e.g., from about 100 mg to about 3grams nucleic acid), fractions will typically be at least 50 ml to 2liters where the nucleic acid peak is expected, with increases in volumein the fractions past the expected peak. Analytical determinations ofnucleic acid yield and purity are performed on single or multiplefractions. In addition, Limulus ameobocyte lysate (LAL) assays (e.g., todetect the Lipid A portion of endotoxin) may be performed on eachfraction to determine residual endotoxin and/or other assays describedherein may be performed on each fraction to determine residualpolysaccharide contaminant levels, such as colanic acid or relatedpolysaccharides (as described below), in each fraction. Fractionscontaining high levels of nucleic acid and low endotoxin or low colanicacid may be pooled, or maintained as separate fractions. The resultingnucleic acid samples may again be filtered (e.g., through a 0.2 μmfilter) or subjected to further chromatography techniques depending onthe endotoxin and polysaccharide levels and the desired purity, asdescribed below.

The support matrices for the ion exchange chromatographic materialsdisclosed herein are not critical, however, support matrices based ondextran, cellulose, cross-linked agarose, synthetic organic polymers,coated silica or agarose are conventional in the art and suitable foruse herein.

Affinity Chromatography

As noted above, affinity chromatography can additionally oralternatively be employed to purify the biological sample. A particularaspect of the invention involves the colanic acid degradation processusing the polypeptides described herein, and combining the digestedmaterial with an affinity chromatography material to selectively removedigested polysaccharides such as colanic acid. Particularly preferredaffinity chromatography materials have an affinity for vicinal orcis-diols. In one particular embodiment, the affinity chromatographymaterial is a boronate chromatography resin, such as a boronic acid- orboronate-based resin.

In basic respects, the affinity chromatography involves preparation of aselective adsorbent by covalent immobilization of a molecule, containinga recognizable region, for which the target sample to be separated isspecific, to a suitable insoluble support. The immobilized compound isgenerally referred to as a ligand and it is recognized that coupling ofthe ligand to the support must be accomplished in a manner which doesnot interfere with its ability to be recognized by the target. Affinitybetween ligand and the target molecule to be purified can beaccomplished by passing the sample containing a sample containing thetarget molecule through a column containing the selective adsorbent.Purification is thereafter accomplished by washing the column with abuffer used to free the adsorbent matrix of unwanted materials, followedby elution of the adsorbed target molecule. Washing is accomplished bypassing a volume of physiological buffer, such as phosphate bufferedsaline, about pH 7.2, through the column. The volume of buffer used inthe washing step should not be so great as to result in target moleculeloss but, on the other hand, not so limited so as not to removeimpurities. Elution is the step wherein the target molecule is removedfrom the column by using a solvent that reduces the affinity of thetarget molecule to the ligand or the affinity of the ligand-targetmolecule complex to the solid support. Elution of an antibody coupled tothe antigen may be accomplished by either a salt gradient, to change thepH; buffered step-gradient, to change the ionic strength; or othermethods.

The ideal solid support or matrix should possess several characteristicsincluding, macroporosity, mechanical stability, ease of activation,hydrophilicity, and inertness, i.e., low nonspecific adsorption. Nomatrix is ideal in all of these respects; the matrix is often determinedempirically. Affinity chromatography matrices commonly used by thoseskilled in the art include cross-linked dextran, agarose,polyacrylamide, cellulose, silica and poly(hydroxyethylmethacrylate).For immuno-adsorbents, beaded agarose is generally the preferred solidsupport by those skilled in the art due to its high adsorptive capacityfor proteins, high porosity, hydrophilicity, chemical stability, lack ofcharge and relative inertness toward nonspecific adsorption.

Ligands may be physically adsorbed to matrices or covalently attached topolymeric matrices containing hydroxylic or amino groups by means ofbifunctional reagents. Attachment usually requires two steps, activationof the matrix and coupling of the ligand to the activated matrix.Activated matrices are available commercially. The selection method forcoupling the ligand to the matrix is dictated in part by the choice ofmatrix, and, in part, by the choice of ligand.

The specific buffering conditions used for equilibrating the affinitycolumn in preparation for sample application should reflect the specificproperties of the interacting system being used. The nature of thebuffer used, including its pH and ionic strength, should be optimal forthe ligand-target molecule system. The target sample applied to thecolumn should preferably be contained in the same buffer used toequilibrate the column. After sample application and adsorption, thecolumn may be washed with the starting buffer to remove any unboundsample and any impurities. It is also common to then wash the columnwith buffers different from the starting buffer in order to removenonspecifically adsorbed substances.

Elution of the target molecule may be accomplished by a number ofmethods, including but not limited to these presented here. Typically,the conditions of the buffer may be changed such that the affinity ofthe binding complex falls sufficiently, thereby destroying effectivebinding to each other or to the solid support. This is achieved byaltering the pH, or the ionic strength of the buffer or both, or bychaotropic ions, e.g., cyanates. Increased separation may be obtained bygradient elution. Suitable methods of elution include use of chaotropicagents such as KSCN; organic solvents, e.g., ethylene glycol, DMSO, oracetonitrile; denaturing agents, e.g., 8 M urea or 6 M guanine;electrophoretic elution; pressure induced elution and metal ion elution.Incomplete elution results in both loss of product and loss of columncapacity. Ideally, the elution conditions should allow for completeelution of the product after one or two column volumes have passedthrough the column.

Detailed discussions of affinity chromatography can be found in Handbookof Affinity Chromatography, David S. Hage (ed.) CRC Press (2006), andAffinity Chromatography: A Practical Approach, edited by Dean P. D. G.,Johnson, W. S., Middle, F. A., Affinity Chromatography, Principles andMethods, as published by Pharmacia, (Pharmacia LKB Biotechnology,Uppsala, Sweden), and Immunoaffinity Purification: Basic Principles andOperational Considerations, Yarmush, M. L, et al., (1992) Biotech Adv.,10:412-446.

Ligands used for affinity chromatography are structurally andbiologically closely related to the target molecule to be purified. Forthe purposes of the present invention, any suitable affinitychromatography material and ligand thereof can be employed for bindingand eluting the sample of interest (i.e., plasmid DNA). In general, thismakes the selection of the ligand specific for each case.

Preferably, the ligand used in the affinity chromatography material hasspecificity for diol complexes, preferably vicinal or cis-diols.Polysaccharides, such as colanic acid, for example, contain vicinal diolcomplexes. In a particularly preferred embodiment, the affinitychromatography material is a boronate affinity chromatography material.Boronate affinity columns were first employed for the separation ofsugars and nucleic acid components by Weith et al, Biochemistry 9,4396-4401, 1970; since then, this technique has been employed in theseparation of a wide range of cis-diol compounds, including nucleosides,nucleotides, carbohydrates, glycoproteins, and enzymes.

In general, the mechanism of action between boronic acids and cis-diolsinvolves hydroxylation of the boronate under basic conditions; theboronate goes from a trigonal coplanar form to a tetrahedral boronateanion, which can then form esters with cis-diols. The resulting diestercan by hydrolyzed under acidic conditions, thus reversing the reaction.Other methods for separation of vicinal diols are described by Barry etal., Australian J. Chem. 37, (1984); Gable, Organometallics 13(6),2486-88 (1994); Liu, J. Microbial Methods 29, 85-95 (1997) Kinrade etal., DaltonTrans. 3713-3716 (2003); and Zhao et al., AnalyticalSciences, 22(5), 747 (2006).

Suitable boronate ligands for use in the affinity chromatographymaterial of the present processes include, for example,3-aminophenylboronic acid (3aPBA),2-(((4-boronophenyl)-methyl)-ethylammonio)ethyl,2-(((4-boronophenyl)-methyl)-diethylammonio)ethyl,p-(ω-aminoethyl)phenyl-boronate, poly(p-vinylbenzeneboronic acid),N-(4-nitro-3-dihydroxyborylphenyl)succinamic acid,4-(N-methyl)carboxamido-benzeneboronic acid),3-nitro-4-carboxamidobenzeneboronic acid,2-nitro-3-succinamido-benzeneboronic acid, and3-succinamido-4-nitro-benzeneboronic acid, among others. One preferredboronate ligand is 3-aminophenylboronic acid (3aPBA).

A range of support matrices for the affinity chromatographic materialsdisclosed herein, including boronate affinity chromatographic materials,are not critical, however, support matrices based on dextran, cellulose,agarose, polyacrylamide, silica, polystyrene, and polymethacrylate areconventional in the art and suitable for use herein. Boronate affinitymatrices are commercially available from a variety of vendors, includingfor example, Sigma-Aldrich Inc. (Boric acid gel; polymethacrylatesupport) (m-aminophenylboronic acid-acrylate; acrylic bead support);Bio-Rad (AFFI-GEL 601™; polyacrylamide support); Pierce (immobilizedboronic acid gel; polyacrylamide support); and Tosoh(m-aminophenylboronic acid-agarose; agarose support) (TSK-GEL™Boronate-5PW column; polymethacrylate support). Other companiessupplying boronic acid and derivatives thereof include Denisco(Hyderabad, India) and Synthonix Corporation (Wake Forest, N.C.).

Polymer gels containing boronate groups described in U.S. Pat. No.5,969,129 (hereby incorporated by reference herein in its entirety) mayalso be used.

The principles, theory and devices used for boronate affinitychromatography are also described in Boronate Affinity Chromatography,Chapter 8, pages 215-230, Handbook of Affinity Chromatography, David S.Hage (ed.) CRC Press (2006).

Hydrophobic Interaction Chromatography

In the embodiments in which hydrophobic interaction chromatography (HIC)materials and methods are employed, these chromatography methodsgenerally employ hydrophobic moieties on a substrate to attracthydrophobic regions in molecules in the sample for purification. Ingeneral, HIC supports work by a clustering effect; typically, nocovalent or ionic bonds are formed or shared when these moleculesassociate. Hydrophobic interaction chromatography is beneficial as it isat least partially removes open circular plasmid forms and othercontaminants, such as genomic DNA, RNA, and endotoxin.

For the purposes of the present invention, any suitable hydrophobicinteraction matrix can be employed for binding and eluting the sample ofinterest (e.g., plasmid DNA). Such hydrophobic interaction matricesinclude, but are not limited to, natural or artificial surfacescontaining uncharged groups, such as methyl, ethyl, or other alkylgroups. These groups form hydrophobic bonds with proteins which arepassed through the matrix and result in separation of polynucleotidesand/or polypeptides based on the strength of interaction between thepolynucleotides and/or polypeptides and matrix groups. The degree ofhydrophobicity of the resin material may vary depending on theconcentration of salt in the medium or the concentration of salt in theeluent. Hydrophobic interaction columns normally comprise a base matrix(e.g., cross-linked agarose or synthetic copolymer material) to whichhydrophobic ligands (e.g., alkyl or aryl groups) are coupled. Preferredhydrophobic interaction chromatography resins generally include alkylmoieties of 2 to 20 carbon atoms in length (e.g., 4 to 18 carbon atoms,or 6 to 15 carbon atoms), which are typically unsubstituted.

The pore diameter of the base material for hydrophobic interactionchromatography is generally between 500 to 4000 Å, but it can beappropriately selected from said range depending on the molecular sizeof sample to be separated and the components thereof. In general, sincethe retention of nucleic acids on the packing material and theadsorption capacity may differ depending on the pore diameter, it may bepreferable to use a base material with a relatively large pore diameterfor nucleic acids with relatively large molecular size and a basematerial with relatively small pore diameter for nucleic acids with arelatively small molecular size.

Hydrophobic interaction chromatography can be performed at low or highpressures, wherein the column is equilibrated in the presence of aqueousbuffers using relatively high salt concentrations (e.g., 1.2 to 1.7 Mammonium sulfate) and eluted in the presence of aqueous buffers usingrelatively low salt concentrations (e.g., a decreasing ammonium sulfategradient from 1.2 M to 0.5 M). As such, polynucleotides and polypeptidesare selectively eluted based on the differing strengths of hydrophobicinteraction with the hydrophobic groups on the matrix, i.e., in order ofincreasing hydrophobicity of the protein. Examples of commerciallyavailable hydrophobic interaction matrices for relatively low pressureapplications include phenyl-SEPHAROSE™ (Pharmacia) and butyl and etherTOYOPEARL 650™ series resins (Toso Haas). Other commercially availablehydrophobic interaction chromatography resins include Phenyl SEPHAROSE 6FAST FLOW™ column with low or high substitution (Pharmacia LKBBiotechnology, AB, Sweden); Phenyl SEPHAROSE™ High Performance column(Pharmacia LKB Biotechnology, AB, Sweden); Octyl SEPHAROSEX HighPerformance column (Pharmacia, LKB Biotechnology, AB, Sweden);FRACTOGEL™ EMD Propyl or FRACTOGEL™ EMD Phenyl columns (E. Merck,Germany); MACRO-PREP™ Methyl or MACRO-PREP™ t-Butyl Supports (Bio-Rad,California); and WP HI-Propyl (C3)™ column (J. T. Baker, New Jersey).Still other commercially available hydrophobic interactionchromatography resins are available from Sigma-Aldrich, Inc. (St. Louis,Mo.) (e.g., TSK-GEL® Butyl-NPR; TSK-GEL® Ether-5PW; TSK-GEL® Phenyl-5PW;each of which and others may have various particle sizes); GE Healthcare(Piscataway, N.J.) (e.g., HI-SCREEN™ Phenyl FF (high or low sub);HI-SCREEN™ Butyl FF; HI-SCREEN™ Butyl-S FF; HI-SCREEN™ Octyl FF).

Elution from the hydrophobic interaction matrix can be performed with astep-wise or linear gradient. Suitable elution buffers are well known inthe art. Suitable column sizes are described above in connection withthe ion exchange chromatography materials. Likewise, the supportmatrices for the hydrophobic interaction chromatographic materialsdisclosed herein are not critical, however, support matrices based ondextran, cellulose, cross-linked agarose, synthetic organic polymers,coated silica or agarose are conventional in the art and suitable foruse herein.

Synthesis of base materials for hydrophobic interaction chromatography,as well as process for preparing, polymerizing and functionalizinghydrophobic interaction chromatography and eluting and separatingsamples such as plasmid DNA therethrough are well known in the art, andare inter alia described in U.S. Pat. No. 6,441,160 and U.S. Pat. No.7,169,917 (each of which is hereby incorporated by reference herein inits entirety).

Filtration

According to certain preferred embodiments, one or more filtration,ultrafiltration, or diafiltration steps may also be performed, includingtangential flow filtration. Filtration through size exclusion filterscan be used to at least partially remove endotoxin and othercontaminants while resulting in minimal nucleic acid loss. For manyapplications, for example, it will be desirable to further purify thesample (e.g., plasmid DNA), lower the salt concentration of theresulting sample, concentrate the sample, and/or exchange the buffer toa more suitable buffer for subsequent uses. For therapeutic uses, e.g.use in gene therapy, it may be desirable to further purify the nucleicacid obtained from the tangential flow filtration, or other, step.

One or more (initial or final) filtration, ultrafiltration, ordiafiltration steps may be performed to generally achieve thatresult(s). If desired, a smaller MWCO ultrafiltration membrane may beused for subsequent or final diafiltration steps than used previouslyfor initial purification, since the nucleic acid will typically behighly purified at later stages and predominantly small solute moleculeswill be passed through the membrane into the filtrate. For many plasmidDNAs, for example, a 10,000 to 100,000 MWCO membrane, or greater may beused. Hollow fiber devices with about a 100,000 MWCO membrane arecommonly used, particularly when handling concentrated nucleic acidsolutions, due to smaller hold-up volumes, increased flux, higher yieldsand shorter processing times. Standard, commercially availablefiltration and diafiltration materials are suitable for use in thisprocess, according to standard techniques known in the art.

Filtration of fine particle size contaminants from fluids has beenaccomplished by the use of various porous filter media through which acontaminated composition is passed such that the filter retains thecontaminant. Retention of the contaminant may occur by mechanicalstraining or electrokinetic particle capture and adsorption. Inmechanical straining, a particle is retained by physical entrapment whenit attempts to pass through a pore smaller than itself. In the case ofelectrokinetic capture mechanisms, the particle collides with a surfacewithin the porous filter and is retained on the surface by short-rangeattractive forces. To achieve electrokinetic capture, charge-modifyingsystems can be used to alter the surface charge characteristics of afilter (see, e.g., WO 90/11814). For example, where the contaminant tobe removed is anionic, a cationic charge modifier can be used to alterthe charge characteristics of the filter such that the contaminant isretained by the filter.

In certain embodiments, either before or after filtration, the sample istreated with an aqueous solution comprising a zwitterionic detergent.Suitable zwittergents include, for example, EMPIGEN BB®(n-dodecyl-N,Ndimethylglycine), ZWITTERGENT® 3-08, ZWITTERGENT® 3-10,ZWITTERGENT® 3-12, ZWITTERGENT® 3-14, ZWITTERGENT® 3-16, CHAPS™,CHAPSO™, and others.

In one preferred embodiment, the sample is filtered using tangentialflow filtration. The principles, theory and devices used for tangentialflow filtration are described in Michaels, S. L. et al., “TangentialFlow Filtration” in Separations Technology, Pharmaceutical andBiotechnology Applications, W. P. Olson, ed., Interpharm Press, Inc.,Buffalo Grove, Ill. (1995). To filter and concentrate a sample bytangential flow filtration, for example, a membrane is generallyselected with a molecular weight cut off (MWCO) that is substantiallylower than the molecular weight of the molecules to be retained. Ageneral rule is to select a membrane with a molecular weight cut offthat is 3 to 6 times lower than the molecular weight of the molecules tobe retained. The membrane is installed, the tangential flow filtrationsystem is initialized (typically flushed with water and tested for waterfiltrate flow rate and integrity), sample is added, a crossflow isestablished, feed and retentate pressures are set, and filtrate iscollected. When the desired concentration or volume is reached, theprocess is stopped, and the sample is recovered.

One preferred filtration method is diafiltration using anultrafiltration membrane having a molecular weight cutoff in the rangeof 30,000 to 500,000 MWCO, depending on the plasmid size. This step ofdiafiltration allows for buffer exchange, followed by concentration. Theeluate is typically concentrated 3- to 4-fold by tangential flowfiltration as described above using, for example, 30 kDa membranecut-off, to a target concentration, and the concentrate is bufferexchanged by diafiltration at constant volume and adjusted to the targetplasmid concentration. The resulting plasmid DNA solution may then befurther filtered, e.g., through a 0.2 μm filter, and is typicallydivided into several aliquots, which are stored in containers at arelatively cold temperature (e.g., ˜0° C.) until further processing.

Additionally or alternatively, the filter may be one which binds nucleicacid while allowing endotoxins and other contaminants to pass throughthe filter. Once the undesirable materials have passed through thefilter, the nucleic acid may be eluted from the filter and collected.

Suitable size exclusion filters are available from a variety ofcommercial sources including, e.g., Ambion (Austin, Tex.), GE Healthcare(Piscataway, N.J.), Gelman (Ann Arbor, Mich.), Pall-Filtron (East Hills,N.Y.), Roche (Basel, Switzerland), Sartorius (Edgewood, N.Y.), andThermo Scientific Pierce (Rockford, Ill.). The filter used will be onethat binds endotoxin and other contaminants while allowing nucleic acidto pass through. Pall Ultipor® N₆₆® filters have been found to removesubstantial endotoxin with high yield of nucleic acid. The lysatesolution or lysate derivative containing the nucleic acid may also bepre-filtered (e.g., using a 0.45 μm filter) prior to one or more of thechromatography or filtration steps described herein.

Where DNA purified according to the above protocol is to be complexedwith a lipid carrier for use in gene therapy, for example, it may alsobe desirable to exchange the DNA into a low conductivity buffer,preferably by diafiltration. A low-conductivity buffer is meant toinclude any buffer of less than about 10 mS, preferably less than about1 mS.

In addition to the filtration techniques described above, conventionalgel filtration methods (i.e., using a size exclusion chromatographymaterial) may be employed. See, e.g., Gel Filtration Principles andMethods, Edition AI, Amersham Biosciences, 2002.

The polypeptides described herein may additionally or alternatively usedin a variety of industrial processes requiring or benefiting from thedigestion of colanic acid and other polysaccharides. In general, thismay involve treating machinery, product and/or effluent pipelines, andthe products, intermediates, or effluents themselves with thepolypeptides described herein in order to, for instance, remove orprevent corrosion, fouling, or other build-up resulting from theseindustrial processes. One particular example is the removal orprevention of biofilms (e.g., those containing bacteria, such as gramnegative bacterial) that may form during the processes or over time;biofilms and similar materials can constrict or block passageways andconduits, or increase wear-and-tear on machinery parts or systems.Representative industrial processes that may benefit from use of thepolypeptides described herein include, for example, in paper andcellulose-making processes, membrane reconstitution and cleaning,recycling, waste-water treatments (e.g., digestions of aerobic solidsand sludges), petrochemical refining and waste remediation, high puritywater filtration and systems, water cooling systems/heat exchangers, andfood processing. For paper or cellulose processing operations, forexample, biofilms may form in intermediate processing streams, which canadversely influence downstream processing and/or affect the quality ofthe final product. The polypeptides described herein may benefit suchprocesses by removing, minimizing, or preventing such biofilms.

Polypeptides

As noted above, the present invention relates to polypeptides that arecapable of degrading colanic acid that may be found, for example, inbiological materials. This may include, for example, biofilms orbacterial nucleic acid preparations derived from bacteria such as E.coli, Salmonella, or other enterobacteriaceae, such as plasmid DNAderived from E. coli. The polypeptides described herein alsoadvantageously enable the preparation of highly pure gram negativebacterial plasmid DNA preparations.

In certain embodiments, the polypeptide comprises an amino acid sequencegenerally corresponding to SEQ ID NO: 1, and conservative amino acidsubstitutions thereof. This polypeptide generally corresponds to afull-length colanic acid-degrading polypeptide having a molecular weightof about 84,354 Daltons. In general, the polypeptide is an isolatedpolypeptide. In certain embodiments, the polypeptide is isolated from abacterial source organism and purified. Typically, the polypeptide has apurity of at least 70%, more preferably at least 80%, more preferably atleast 90%, more preferably at least 95%, more preferably at least 98%,and more preferably at least 99%.

Polypeptide fragments are also provided herein. Such fragments may betruncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full-length protein. Forexample, certain fragments lack amino acid residues that are notessential for a desired activity (e.g., biological or otherwise) of thepolypeptide. In one particular, embodiment, the polypeptide comprises anamino acid sequence generally corresponding to SEQ ID NO: 2, andconservative amino acid substitutions thereof. This polypeptidegenerally corresponds to a truncated version of the full-lengthpolypeptide, wherein the first 106 amino acids of the full-lengthpolypeptide (SEQ ID NO: 1) are absent.

In addition to the full-length and truncated polypeptides describedherein, it is contemplated that polypeptide variants can be prepared,e.g., by introducing appropriate nucleotide changes to the polypeptideDNA, and/or by synthesis of the desired polypeptide, or by isolating andpurifying a variant polypeptide having colanic acid-degrading activity.Those skilled in the art will appreciate that amino acid changes mayalter certain post-translational processes of the polypeptide, such aschanging the number or position of glycosylation sites or alteringmembrane anchoring characteristics.

Variations in the full-length and/or truncated sequences or in variousdomains of the polypeptides described herein, can be made, for example,using any of the techniques and guidelines for conservative andnon-conservative mutations set forth in the literature (for example,U.S. Pat. No. 5,364,934 (hereby incorporated by reference herein in itsentirety)). Variations may be a substitution, deletion or insertion ofone or more codons encoding the polypeptide that results in a change inthe amino acid sequence of the polypeptide as compared with the nativesequence polypeptide. Optionally the variation is by substitution of atleast one amino acid with any other amino acid in one or more of thedomains of the polypeptide. Guidance in determining which amino acidresidue may be inserted, substituted or deleted without adverselyaffecting the desired activity may be found by comparing the sequence ofthe polypeptide with that of homologous protein molecules and minimizingthe number of amino acid sequence changes made in regions of highhomology. Amino acid substitutions can be the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with a serine,i.e., conservative amino acid replacements. Insertions or deletions mayoptionally be in the range of about 1 to 5 amino acids, 5 to 10 aminoacids, 10 to 25 amino acids, 25 to 50 amino acids, or more, such as 100amino acids or more. The variation allowed may be determined bysystematically making insertions, deletions or substitutions of aminoacids in the sequence and testing the resulting variants for activityexhibited by the full-length or mature native sequence.

As noted above, the polypeptide may be a variant of the full-length ortruncated polypeptide described herein. Ordinarily, a polypeptidevariant will have at least about 80% amino acid sequence identity,alternatively at least about 81% amino acid sequence identity,alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to afull-length polypeptide sequence as disclosed herein (e.g., SEQ ID NO:1).

For truncated polypeptide variants, the polypeptide variant willordinarily have at least about 80% amino acid sequence identity,alternatively at least about 81% amino acid sequence identity,alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to atruncated polypeptide sequence as disclosed herein (e.g., SEQ ID NO: 2).

In one embodiment, the polypeptide comprises an amino acid sequencehaving at least about 90% amino acid sequence identity to SEQ ID NO: 1,and conservative amino acid substitutions thereof. In anotherembodiment, the polypeptide comprises an amino acid sequence having atleast about 95% amino acid sequence identity to SEQ ID NO: 1, andconservative amino acid substitutions thereof. In another embodiment,the polypeptide comprises an amino acid sequence having at least about98% amino acid sequence identity to SEQ ID NO: 1, and conservative aminoacid substitutions thereof. In another embodiment, the polypeptidecomprises an amino acid sequence having at least about 99% amino acidsequence identity to SEQ ID NO: 1, and conservative amino acidsubstitutions thereof.

In one embodiment, the polypeptide comprises an amino acid sequencehaving at least about 90% amino acid sequence identity to SEQ ID NO: 2,and conservative amino acid substitutions thereof. In anotherembodiment, the polypeptide comprises an amino acid sequence having atleast about 95% amino acid sequence identity to SEQ ID NO: 2, andconservative amino acid substitutions thereof. In another embodiment,the polypeptide comprises an amino acid sequence having at least about98% amino acid sequence identity to SEQ ID NO: 2, and conservative aminoacid substitutions thereof. In another embodiment, the polypeptidecomprises an amino acid sequence having at least about 99% amino acidsequence identity to SEQ ID NO: 2, and conservative amino acidsubstitutions thereof.

In certain embodiments, exemplary conservative substitutions of interestare shown in Table 1. If such substitutions result in a change inbiological activity, or a reduction in the desired activity, then moreother changes, such as those described below in reference to amino acidclasses, may be introduced and the products screened. It is understoodthat codons capable of coding for such conservative substitutions areknown in the art.

TABLE 1 Original Residue Conservative Substitutions Ala (A) Ser; Val;Leu; Ile Arg (R) Lys; Gln; Asn Asn (N) Gln; His; Lys; Arg Asp (D) GluCys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp Gly (G) Pro; Ala His (H) Asn;Gln; Lys; Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu (L) Ile;Val; Met; Ala; Phe; Norleucine Lys (K) Arg; Gln; Glu; Asn Met (M) Leu;Ile; Phe Phe (F) Leu; Val; Ile; Ala; Met; Tyr Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe; Thr; Ser Val (V) Ile;Leu; Met; Phe; Ala; Norleucine

Within the scope of the present invention is polypeptide analogs of theinvention arrived at by amino acid substitutions based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, etc. One factor that canbe considered in making amino acid substitutions is the hydropathicindex of amino acids. The importance of the hydropathic amino acid indexin conferring interactive biological function on a protein has beendiscussed by Kyte and Doolittle (J. Mol. Biol., 157: 105-132, 1982). Itis accepted that the relative hydropathic character of amino acidscontributes to the secondary structure of the resultant protein. This,in turn, affects the interaction of the protein with molecules such asenzymes, substrates, receptors, DNA, antibodies, antigens, etc.

Based on its hydrophobicity and charge characteristics, each amino acidhas been assigned a hydropathic index as follows: isoleucine (+4.5);valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine(+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine(−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline(−1.6); histidine (−3.2); glutamate/glutamine/aspartate/asparagine(−3.5); lysine (−3.9); and arginine (−4.5).

As is known in the art, certain amino acids in a peptide or protein canbe substituted for other amino acids having a similar hydropathic indexor score and produce a resultant peptide or protein having similarbiological activity, i.e., which still retains biological functionality.In making such changes, it is preferable that amino acids havinghydropathic indices within ±2 are substituted for one another. Morepreferred substitutions are those wherein the amino acids havehydropathic indices within ±1. Most preferred substitutions are thosewherein the amino acids have hydropathic indices within ±0.5.

Like amino acids can also be substituted on the basis of hydrophilicity.U.S. Pat. No. 4,554,101 discloses that the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with a biological property of theprotein. The following hydrophilicity values have been assigned to aminoacids: arginine/lysine (+3.0); aspartate/glutamate (+3.0±1); serine(+0.3); asparagine/glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine/histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine/isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5); and tryptophan (−3.4). Thus, one amino acid in apeptide, polypeptide, or protein can be substituted by another aminoacid having a similar hydrophilicity score and still produce a resultantprotein having similar biological activity, i.e., still retainingcorrect biological function. In making such changes, amino acids havinghydropathic indices within ±2 are preferably substituted for oneanother, those within ±1 are more preferred, and those within ±0.5 aremost preferred.

Substantial or minor modifications in function or biological or otheridentity of the polypeptides of the invention are also accomplished byselecting substitutions that differ significantly in their effect onmaintaining, among other things: (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation; (b) the charge or hydrophobicity of the moleculeat the target site; or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2)neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic:asn, gln, his, lys, arg; (5) residues that influence chain orientation:gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will generally entail exchanging a memberof one of these classes for another class. Such substituted residuesalso may be introduced into the conservative substitution sites or, morepreferably, into the remaining (non-conserved) sites.

In one embodiment, for example, in the amino acid sequences described inSEQ ID NO: 1, 2, 3, 4, 5, and/or 6, the amino acid leucine (L) mayalternatively be either leucine (L) or isoleucine (I), the amino acidaspartic acid (D) may alternatively be aspartic acid (D) or asparagine(N), the amino acid glutamine (Q) may alternatively be glutamine (Q) orlysine (K), and the amino acid phenylalanine (F) may alternatively bephenylalanine (F) or oxidized methionine.

The variations can be made using methods known in the art, such asalanine scanning, oligonucleotide-mediated (site-directed) mutagenesis,and PCR mutagenesis, among other known techniques. Site-directedmutagenesis, for example, (see, e.g., Carter et al., Nucl. Acids Res.,13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)),cassette mutagenesis (see, e.g., Wells et al., Gene, 34:315 (1985)),restriction selection mutagenesis (see, e.g., Wells et al., Philos.Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniquescan be performed on cloned DNA to produce the CAE variant DNA. Scanningamino acid analysis can also be employed to identify one or more aminoacids along a contiguous sequence. Among the preferred scanning aminoacids are relatively small, neutral amino acids. Such amino acidsinclude alanine, glycine, serine, and cysteine. Alanine is typically apreferred scanning amino acid among this group because it eliminates theside-chain beyond the beta-carbon and is less likely to alter themain-chain conformation of the variant (see, e.g., Cunningham and Wells,Science, 244: 1081 1085 (1989)). Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions (see Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). Ifalanine substitution does not yield adequate amounts of variant, or thecolanic acid-degrading ability of the resulting polypeptide isdiminished or non-existent, another amino acid can be used.

Covalent modifications of the polypeptides described herein are alsoincluded within the scope of this invention. One type of covalentmodification, for example, includes reacting targeted amino acidresidues of a polypeptide with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues of the polypeptide. Derivatization with bifunctional agents isuseful, for instance, for crosslinking the polypeptide(s) to awater-insoluble support matrix or surface for use in the method forpurifying anti-CAE antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate. In one particularembodiment, the C-terminal isoleucine of certain polypeptides describedherein can be removed or deleted to expose a terminal tyrosine which canbe used, for example, to crosslink the polypeptide to an insolublematrix, either directly or through a spacer, to form an affinity resinor immobilized resin.

Other modifications include, for instance, deamidation of glutaminyl andasparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively, hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and/or histidineside chains (see, e.g., T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79 86(1983)), acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group.

Another type of covalent modification of the polypeptides describedherein and included within the scope of this invention comprisesaltering the native glycosylation pattern of the polypeptide. Ingeneral, altering the native glycosylation pattern involves deleting oneor more carbohydrate moieties found in the polypeptide (e.g., thefull-length) sequence (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the polypeptide sequence. In addition, this may includequalitative changes in the glycosylation of the native proteins,involving a corresponding change in the nature and proportions of thevarious carbohydrate moieties that may be present.

Addition of glycosylation sites to the polypeptide of the invention maybe accomplished by altering the amino acid sequence. The alteration maybe made, for example, by the addition of, or substitution by, one ormore serine or threonine residues to the polypeptide sequence (forO-linked glycosylation sites). The amino acid sequences described herein(e.g., SEQ ID NO. 1, SEQ ID NO. 2, etc.) may optionally be alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thepolypeptide is by chemical or enzymatic coupling of glycosides to thepolypeptide. Such methods are generally described in the art, e.g., inPCT International Pub. No. WO 87/05330, and in Aplin and Wriston, CRCCrit. Rev. Biochem., pp. 259 306 (1981).

Removal of carbohydrate moieties present on the polypeptides describedherein may be accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification comprises linking the polypeptidesdescribed herein to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337 (each of which is hereby incorporatedby reference herein).

Additionally or alternatively, the polypeptides of the present inventionmay also be modified in a way to form a chimeric molecule comprising thepolypeptide fused to another, heterologous polypeptide or amino acidsequence. The polypeptides may also be labeled with reagents thatfacilitate their detection. For example, the agents may be combined withfluorescent labels (e.g., Prober et al., Science 238:336-340 (1987);Albarella et al., EP 0 144 914); chemical labels (e.g., Sheldon et al.,U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No. 4,563,417);and/or modified bases (e.g., Miyoshi et al., EP 0 119 448) (each ofwhich are hereby incorporated by reference in their entirety).

In one embodiment, such a chimeric molecule comprises a fusion of thepolypeptide with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the polypeptide amino acidsequence. The presence of such epitope-tagged forms of the polypeptidecan be detected using an antibody against the tag polypeptide. Also,provision of the epitope tag enables the polypeptide to be readilypurified by affinity purification using an anti-tag antibody or anothertype of affinity matrix that binds to the epitope tag. Various tagpolypeptides and their respective antibodies are well known in the art.Examples include the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (see, e.g., Evan et al., Molecular and CellularBiology, 5:3610 3616 (1985)); the flu HA tag polypeptide and itsantibody 12 CA5 (see, e.g., Field et al., Mol. Cell. Biol., 8:2159 2165(1988)); the Herpes Simplex virus glycoprotein D (gD) tag and itsantibody (see, e.g., Paborsky et al., Protein Engineering, 3(6):547 553(1990)); and poly-histidine (poly-his) or poly-histidine-glycine(poly-his-gly) tags. Other tag polypeptides include an α-tubulin epitopepeptide (see, e.g., Skinner et al., J. Biol. Chem., 266:15163 15166(1991)); the FLAG®-peptide (Sigma-Aldrich, Inc. (St. Louis, Mo.); seealso Hopp et al., BioTechnology, 6:1204 1210 (1988)); the KT3 epitopepeptide (see, e.g., Martin et al., Science, 255:192 194 (1992)); and theT7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl.Acad. Sci. USA, 87:6393 6397 (1990)).

To facilitate isolation and/or purification, for example, an amino acidtag can be added to the polypeptides described herein using geneticengineering techniques that are well known to practitioners of the art.In certain embodiments, for example, the polypeptide(s) may include one,and more preferably six, consecutive histidine residues at either theamino or carboxy terminus of the protein. Such consecutive histidineresidues are commonly referred to as a histidine tag. Terminalconsecutive histidine residues can facilitate detection and/orpurification of expressed recombinant proteins, and generally do notinterfere with the function/activity/structure of the protein. Theconsecutive histidine residues can be incorporated into the proteincoding gene by primers that carry the 5′-CAT-3′ triplets. Consecutivehistidine residues at either terminus serve as convenient aids forpurification of proteins with immobilized metal affinity chromatography,which exploits the ability of the amino acid histidine to bind chelatedtransition metal ions such as nickel (Ni²⁺), zinc (Zn²⁺) and copper(Cu²⁺). As noted above, other techniques include, but are not limitedto, epitopes for polyclonal or monoclonal antibodies including but notlimited to the T7 epitope, the myc epitope, and the V5a epitope; andfusion of the polypeptides described herein to suitable protein partnersincluding but not limited to glutathione-S-transferase or maltosebinding protein. In a particular embodiment, the amino acid sequencecomprises an affinity tag allowing for, e.g., isolation and purificationof the protein, such as, for example, a GST tag, a His tag, a FLAG® tag,or an XPRESS™ tag; in certain preferred embodiments, the affinity tagcomprises a His tag (i.e., one or more, and preferably six histidineresidues) (see, e.g., SEQ ID NO: 3 or SEQ ID NO: 4), one or more copiesof the FLAG® octapeptide (SEQ ID NO: 18 DYKDDDDK) (see, e.g., SEQ ID NO:5), or the XPRESS™ octapeptide (SEQ ID NO: 19 DLYDDDK). These additionalamino acid sequences can be added to the C-terminus of the polypeptides,as well as the N-terminus, or at intervening positions within thepolypeptides.

In an alternative embodiment, the chimeric molecule may comprise afusion of the polypeptide with an immunoglobulin or a particular regionof an immunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a polypeptide in place of at least one variable region within anIg molecule. In a particular embodiment, the immunoglobulin fusionincludes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regionsof an IgG1 molecule. For the production of immunoglobulin fusions seealso U.S. Pat. No. 5,428,130; U.S. Pat. No. 6,165,476; U.S. Pat. No.6,444,792; U.S. Pat. No. 7,442,778; and U.S. Pat. No. 7,465,447 (each ofwhich is hereby incorporated by reference herein in its entirety).

Preparation of Polypeptides

Full-length polypeptides (including, for example, the polypeptidecomprising the amino acid sequence corresponding to SEQ ID NO: 1) andpolypeptide fragments (including, for example, the truncated polypeptidecomprising the amino acid sequence corresponding to SEQ ID NO: 2) may beprepared by any of a number of conventional techniques. The polypeptidesof the invention may be prepared, in general, by culturing host cellstransformed or transfected with a vector containing polynucleotidesencoding the desired polypeptide. In certain embodiments, the vector isselected from a plasmid, a virus, and a bacteriophage; more preferablyin this embodiment, the vector is a bacteriophage (see, e.g., Example2). Methods of preparing vectors, and in particular phage, for theproduction of polypeptides are well known in the art.

Generally, host cells, such as bacteria, are transfected or transformedwith expression or cloning vectors described herein for polypeptideproduction and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. The cultureconditions, such as media, temperature, pH and the like, can be selectedby the skilled artisan without undue experimentation. In general,principles, protocols, and practical techniques for maximizing theproductivity of cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated, and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation,for example, is generally used for prokaryotes. Infection withAgrobacterium tumefaciens is used for transformation of certain plantcells, as described by Shaw et al., Gene, 23:315 (1983) and PCTInternational Pub. No. WO 89/05859. For mammalian cells without suchcell walls, the calcium phosphate precipitation method of Graham and vander Eb, Virology, 52:456 457 (1978) can be employed. General aspects ofmammalian cell host system transfections have likewise been described inU.S. Pat. No. 4,399,216 (hereby incorporated by reference herein in itsentirety). Transformations into yeast are typically carried outaccording to the method of Van Solingen et al., J. Bact., 130:946 (1977)and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However,other methods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527 537 (1990) and Mansour et al.,Nature, 336:348 352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited Enterobacteriaceae such asEscherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratiamarcescans, and Shigella, as well as Bacilli such as B. subtilis and B.licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710),Pseudomonas such as P. aeruginosa, and Streptomyces. Preferably, thehost cell secretes minimal amounts of proteolytic enzymes. For example,the strain may be modified to effect a genetic mutation in the genesencoding proteins endogenous to the host (see, e.g., U.S. Pat. No.4,946,783). Alternatively, in vitro methods of cloning, e.g., PCR orother nucleic acid polymerase reactions, are suitable.

Preferred host cells for producing polypeptides of the invention areprokaryotes, and more preferably bacteria, including eubacteria andarchaebacteria. Preferred of these are eubacteria, includinggram-positive and gram-negative bacteria. More preferred aregram-negative bacteria. One preferred type of bacteria isEnterobacteriaceae. Examples of bacteria belonging to Enterobacteriaceaeinclude Escherichia, Enterobacter, Erwinia, Klebsiella, Proteus,Salmonella, Serratia, and Shigella. Other types of suitable bacteriainclude Azotobacter, Pseudomonas, Rhizobia, Vitreoscilla, andParacoccus. E. coli is particularly preferred herein.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells, including the media generally described by Sambrook et al.,supra. Media that are suitable for bacteria include, but are not limitedto, AP5 medium, nutrient broth, Luria-Bertani (LB) broth, Neidhardt'sminimal medium, and C.R.A.P. minimal or complete medium (see, e.g., U.S.Pat. No. 6,828,121), plus necessary nutrient supplements. The media mayalso contains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene. Anynecessary supplements besides carbon, nitrogen, and inorganic phosphatesources may also be included at appropriate concentrations introducedalone or as a mixture with another supplement or medium such as acomplex nitrogen source. The culture medium may also optionally containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol, anddithiothreitol. The prokaryotic host cells are cultured at suitabletemperatures. For E. coli growth, for example, the preferred temperatureranges from about 20° C. to about 39° C., more preferably from about 25°C. to about 37° C., even more preferably at about 30° C. Any necessarysupplements may also be included at appropriate concentrations thatwould be known to those skilled in the art, introduced alone or as amixture with another supplement or medium such as a complex nitrogensource. The pH of the medium may be any pH from about 5 9, dependingmainly on the host organism. For E. coli, the pH is preferably fromabout 6.8 to about 7.4, and more preferably about 7.0.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forpolypeptide-encoding vectors. Saccharomyces cerevisiae is a commonlyused lower eukaryotic host microorganism. Other conventional hostmicroorganisms include filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357), and Aspergillus hosts such asA. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284289 (1983); Tilburn et al., Gene, 26:205 221 (1983); Yelton et al.,Proc. Natl. Acad. Sci. USA, 81: 1470 1474 (1984)) and A. niger (Kellyand Hynes, EMBO J., 4:475 479 (1985)); Kluyveromyces hosts (U.S. Pat.No. 4,943,529; Fleer et al., Bio/Technology, 9:968 975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 154(2):737 742 (1983)), K. fragilis (ATCC 12,424), K.bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus;yarrowia (EP 0 402 226); Pichia pastoris (EP 0 183 070; Sreekrishna etal., J. Basic Microbiol., 28:265 278 (1988)); Candida; Trichodermareesia (EP 0 244 234); Neurospora crassa (Case et al., Proc. Natl. Acad.Sci. USA, 76:5259 5263 (1979)); Schizosaccharomyces pombe (Beach andNurse, Nature, 290: 140 (1981); EP 0 139 383); and Schwanniomyces suchas Schwanniomyces occidentalis (EP 0 394 538).

Methylotropic yeasts are also suitable and include, but are not limitedto, yeast capable of growth on methanol selected from the generaconsisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces,Torulopsis, and Rhodotorula. Representative species that are exemplaryof this class of yeasts may be found in C. Anthony, The Biochemistry ofMethylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated polypeptides aregenerally derived from multicellular organisms. Examples of invertebratecells include insect cells such as Drosophila S2 and Spodoptera Sf9, aswell as plant cells. Examples of useful mammalian host cell linesinclude Chinese hamster ovary (CHO) and COS cells. More specificexamples include Chinese hamster ovary cells/-DHFR (SEQ ID NO: 20) (CHO,Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); humanliver cells (Hep G2, HB 8065); human lung cells (W138, ATCC CCL 75);monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651); mousesertoli cells (TM4, Mather, Biol. Reprod., 23:243 251 (1980)); and mousemammary tumor (MMT 060562, ATCC CCL51), among others. The selection ofthe appropriate host cell is deemed to be within the skill in the art.

Once identified, the nucleic acid (e.g., cDNA or genomic DNA) encodingthe target polypeptide may be inserted into a replicable vector forcloning (amplification of the DNA) or for expression, and variousvectors are publicly available. The vector may, for example, be in theform of a cosmid, plasmid, phage, or viral particle. Many vectors areavailable for this purpose, and selection of the appropriate vector willdepend mainly on the size of the nucleic acid to be inserted into thevector and the particular host cell to be transformed with the vector.The appropriate nucleic acid sequence (such as described below may beinserted into the vector by a variety of procedures. In general, DNA isinserted into an appropriate restriction endonuclease site(s) usingtechniques known in the art. Vector components generally include, butare not limited to, one or more of a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence. Construction of suitablevectors containing one or more of these components employs standardligation techniques which are known to the skilled artisan.

The polypeptide may be produced recombinantly not only directly, butalso as a fusion polypeptide with a heterologous polypeptide, which maybe a signal sequence or other polypeptide having a specific cleavagesite at the N-terminus of the mature protein or polypeptide. In general,the signal sequence may be a component of the vector, or it may be apart of the polypeptide-encoding DNA that is inserted into the vector.The signal sequence may be a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, Ipp,or heat-stable enterotoxin II leaders. For yeast secretion, for example,the signal sequence may be, e.g., the yeast invertase leader, alphafactor leader (including Saccharomyces and Kluyveromyces α-factorleaders, the latter described in U.S. Pat. No. 5,010,182), or acidphosphatase leader, the C. albicans glucoamylase leader (EP 0 362 179),or the signal described in PCT International Pub. No. WO 90/13646. Inmammalian cell expression, mammalian signal sequences may be used todirect secretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors may also contain a selection gene, alsoreferred to in the art as a selectable marker. Typical selection genesencode proteins that: (a) complement auxotrophic deficiencies; (b)confer resistance to antibiotics or other drugs or toxins, e.g.,ampicillin, G418, hygromycin, neomycin, methotrexate, or tetracycline;or (c) supply critical nutrients not available from complex media, e.g.,the gene encoding D-alanine racemase for Bacilli.

One example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up the colanicacid-degrading polypeptide-encoding nucleic acid, such as DHFR orthymidine kinase. An appropriate host cell when wild-type DHFR isemployed is the CHO cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA,77:4216 (1980). A suitable selection gene for use in yeast is the trp1gene present in the yeast plasmid YRp7 (see, e.g., Stinchcomb et al.,Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper etal., Gene, 10:157 (1980). The trp1 gene provides a selection marker fora mutant strain of yeast lacking the ability to grow in tryptophan, forexample, ATCC No. 44076 or PEP4-1 (see, e.g., Jones, Genetics, 85:12(1977)).

Expression and cloning vectors usually contain a promoter operablylinked to the polypeptide-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includealkaline phosphatase, a tryptophan (trp) promoter system (e.g., Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 0 036 776); the β-lactamase andlactose promoter systems (e.g., Chang et al., Nature, 275:615 (1978);Goeddel et al., Nature, 281:544 (1979)); and hybrid promoters such asthe tac promoter (see, e.g., deBoer et al., Proc. Natl. Acad. Sci. USA,80:21 25 (1983)). Promoters for use in bacterial systems may alsocontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the target polypeptide.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem., 255:2073 (1980)) or other glycolytic enzymes (Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)), such as enolase, hexokinase, glucokinase, glucose-6-phosphateisomerase, glyceraldehyde-3-phosphate dehydrogenase,phosphofructokinase, phosphoglucose isomerase, 3-phosphoglyceratemutase, pyruvate decarboxylase, pyruvate kinase, and triosephosphateisomerase. Other yeast promoters, which are inducible promoters havingthe additional advantage of transcription controlled by growthconditions, are the promoter regions for acid phosphatase, alcoholdehydrogenase 2, degradative enzymes associated with nitrogenmetabolism, enzymes responsible for maltose and galactose utilization,glyceraldehyde-3-phosphate dehydrogenase, isocytochrome C, andmetallothionein. Suitable vectors and promoters for use in yeastexpression are further described in EP 0 073 657.

Target polypeptide transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as adenovirus (such as Adenovirus 2), avian sarcoma virus,bovine papilloma virus, cytomegalovirus, fowlpox virus (see, e.g., UK2,211,504), hepatitis-B virus, polyoma virus, a retrovirus, and SimianVirus 40 (SV40), or from heterologous mammalian promoters (e.g., theactin promoter or an immunoglobulin promoter), and/or from heat-shockpromoters, provided such promoters are compatible with the host cellsystems.

Transcription of a DNA encoding the target polypeptide by highereukaryotes may be increased in many instances by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,typically about from 10 to 300 bp, that act on a promoter to increaseits transcription. Numerous enhancer sequences are known from mammaliangenes (albumin, α-fetoprotein, elastase, globin, and insulin).Typically, an enhancer from a eukaryotic cell virus will be used.Non-limiting examples include the SV40 enhancer on the late side of thereplication origin (bp 100 270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. The enhancer may be spliced into thevector at a position 5′ or 3′ to the polypeptide coding sequence,typically located at a site 5′ from the promoter.

Expression vectors used in prokaryotic (e.g., bacteria) and/oreukaryotic host cells (yeast, fungi, insect, plant, animal, human, ornucleated cells from other multicellular organisms) will also containsequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of prokaryotic, eukaryotic,or viral DNAs or cDNAs. These regions contain nucleotide segmentstranscribed as polyadenylated fragments in the untranslated portion ofthe mRNA encoding the polypeptide of interest. Still other methods,vectors, and host cells suitable for adaptation to the synthesis ofpolypeptides in recombinant vertebrate cell culture are described inGething et al., Nature, 293:620 625 (1981); Mantei et al., Nature,281:40 46 (1979); EP 0 117 060; and EP 0 117 058.

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (see, e.g., Thomas,Proc. Natl. Acad. Sci. USA, 77:5201 5205 (1980)), dot blotting (DNAanalysis), or in situ hybridization, using an appropriately labeledprobe, based on the sequences provided herein. Alternatively, antibodiesmay be employed that can recognize specific duplexes, including DNAduplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-proteinduplexes. The antibodies in turn may be labeled and the assay may becarried out where the duplex is bound to a surface, so that upon theformation of duplex on the surface, the presence of antibody bound tothe duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a full-lengthsequence polypeptide or against a truncated or fragment peptide based onthe DNA sequences provided herein or against exogenous sequence fused toCAE DNA and encoding a specific antibody epitope.

Forms of the polypeptide may be recovered from culture medium or fromhost cells lysates. If membrane-bound, it can be released from themembrane suing a suitable detergent solution (e.g. TRITON-X™ 100) or byenzymatic cleavage. Cells employed in expression of polypeptides can bedisrupted by various physical or chemical means, such as freeze-thawcycling, sonication, mechanical disruption, or cell lysing agents. Itmay be desired to purify the polypeptide(s) from recombinant cellproteins or polypeptides. Exemplary of suitable purification proceduresare ammonium sulfate precipitation; chromatofocusing; chromatography onsilica or on a cation-exchange resin such as DEAE; ethanolprecipitation; fractionation on an ion-exchange column; gel filtrationusing, for example, SEPHADEX™ G-75; metal chelating columns to bindepitope-tagged forms of the peptide; protein A SEPHAROSE™ columns toremove contaminants such as IgG; reverse phase HPLC; and SDS-PAGE.Various methods of protein purification may be employed and such methodsare known in the art and described, for example, in Deutscher, Methodsin Enzymology, 182 (1990); Scopes, Protein Purification: Principles andPractice, Springer-Verlag, New York (1982). In general, the purificationstep(s) performed will depend, for example, on the nature of theproduction process, the particular polypeptide produced, and thedownstream use(s) of the polypeptide.

Alternatively, for instance, desired peptides and fragments thereof maybe chemically synthesized, or may be extracted from a natural sourceorganism(s). Another alternative approach involves generatingpolypeptides and fragments thereof by enzymatic digestion, e.g., bytreating the protein with an enzyme known to cleave proteins at sitesdefined by particular amino acid residues, or by digesting the DNA withsuitable restriction enzymes and isolating the desired fragment. Yetanother suitable technique involves isolating and amplifying a DNAsequence or fragment encoding a desired polypeptide or polypeptidefragment, by polymerase chain reaction (PCR). Oligonucleotides thatdefine the desired termini of the DNA are employed at the 5′ and 3′primers in the PCR. Where the polypeptide is a polypeptide fragment, thepolypeptide fragment preferably shares at least one biological and/orimmunological activity with the native (i.e., full-length) polypeptidedisclosed herein. In certain instances, the polypeptide fragment mayhave greater activity than the full-length polypeptide, or may otherwisebe optimized or improved relative to the full-length polypeptide.

Other alternative methods, which are well known in the art, may also beemployed to prepare the polypeptides described herein. For instance, thepolypeptide sequence, or portions thereof, may be produced by directpeptide synthesis using solid-phase techniques (see, e.g., Stewart etal., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco,Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149 2154 (1963)). Invitro protein synthesis may be performed using automation, or by manualtechniques. Automated synthesis may be accomplished, for instance, usingan Applied Biosystems Peptide Synthesizer (Foster City, Calif.) inaccordance with the manufacturer's instructions. Various portions of thepolypeptides described herein may be chemically synthesized separatelyand combined using chemical or enzymatic methods to produce thefull-length, truncated, or other variant polypeptides.

Polynucleotides

Another aspect of the disclosure provides polynucleotides and fragmentsthereof, and partial or complete complements thereof, mRNA, and/orcoding sequences, preferably in isolated form, including polynucleotidesencoding a polypeptide or enzyme having colanic acid-degrading (CAE)activity and/or a CAE-related protein and fragments thereof (asdescribed above), DNA, RNA, DNA/RNA hybrid, and related molecules,polynucleotides or oligonucleotides complementary to the polynucleotidesdescribed herein or mRNA sequence or a part thereof, and polynucleotidesor oligonucleotides that hybridize to a CAE-encoding polynucleotide ormRNA of the same.

In certain embodiments, the polynucleotide comprises a nucleic acidsequence generally corresponding to SEQ ID NO: 7, and the complementthereof. In certain other embodiments, the polynucleotide comprises anucleic acid sequence generally corresponding to SEQ ID NO: 8.Preferably, the polynucleotide is an isolated polynucleotide. In otherembodiments, the polynucleotide is a recombinant polynucleotide.

Polynucleotide variants are also provided herein. Polynucleotidevariants may contain one or more substituents, additions, deletions,and/or insertions such that the activity of the polynucleotide is notsubstantially diminished, as described above. The effect on the activityof the polynucleotide may generally be assessed as described herein, orusing conventional methods. Generally, polynucleotide variants have atleast about 80% nucleic acid sequence identity with a nucleotide acidsequence encoding a full-length or truncated polypeptide having colonicacid-degrading activity, as disclosed herein or any other fragment of afull-length or truncated polypeptide sequence as disclosed herein.Variants preferably exhibit at least about 85%, 87%, 88% or 89% identityand more preferably at least about 90%, 92%, 95%, 96%, or 97% identityto a portion of a polynucleotide sequence that encodes a polypeptidehaving endotoxin-degrading capabilities. The percent identity may bereadily determined by comparing sequences of the polynucleotides to thecorresponding portion of the target polynucleotide, using any methodincluding using computer algorithms well known to those having ordinaryskill in the art, such as ALIGN™ or the BLAST™ algorithms (see, e.g.,Altschul, J. Mol. Biol. 219:555-565, 1991; Henikoff and Henikoff, Proc.Natl. Acad. Sci. USA 89:10915-10919, 1992), which is available at theNCB! Website, and which are described elsewhere herein. Defaultparameters may be used.

Ordinarily, a variant polynucleotide will have at least about 80%nucleic acid sequence identity, alternatively at least about 81% nucleicacid sequence identity, alternatively at least about 82% nucleic acidsequence identity, alternatively at least about 83% nucleic acidsequence identity, alternatively at least about 84% nucleic acidsequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity with a nucleic acid sequence encoding a full-lengthpolypeptide sequence as disclosed herein (e.g., SEQ ID NO: 7).

For polynucleotides encoding truncated polypeptides, the polynucleotidevariant will ordinarily have at least about 80% nucleic acid sequenceidentity, alternatively at least about 81% nucleic acid sequenceidentity, alternatively at least about 82% nucleic acid sequenceidentity, alternatively at least about 83% nucleic acid sequenceidentity, alternatively at least about 84% nucleic acid sequenceidentity, alternatively at least about 85% nucleic acid sequenceidentity, alternatively at least about 86% nucleic acid sequenceidentity, alternatively at least about 87% nucleic acid sequenceidentity, alternatively at least about 88% nucleic acid sequenceidentity, alternatively at least about 89% nucleic acid sequenceidentity, alternatively at least about 90% nucleic acid sequenceidentity, alternatively at least about 91% nucleic acid sequenceidentity, alternatively at least about 92% nucleic acid sequenceidentity, alternatively at least about 93% nucleic acid sequenceidentity, alternatively at least about 94% nucleic acid sequenceidentity, alternatively at least about 95% nucleic acid sequenceidentity, alternatively at least about 96% nucleic acid sequenceidentity, alternatively at least about 97% nucleic acid sequenceidentity, alternatively at least about 98% nucleic acid sequenceidentity and alternatively at least about 99% nucleic acid sequenceidentity with a nucleic acid sequence encoding a truncated polypeptidesequence as disclosed herein (e.g., SEQ ID NO: 8).

In one embodiment, the nucleic acid molecule shares at least 90%sequence identity with the nucleic acid sequence set forth in SEQ ID NO:7. For example, the nucleic acid molecule may share at least 95%sequence identity with the nucleic acid sequence set forth in SEQ ID NO:7, or may share at least 98% sequence identity with the nucleic acidsequence set forth in SEQ ID NO: 7. In a particular embodiment, thenucleic acid molecule has the sequence set forth in SEQ ID NO: 7; thatis, the nucleic acid molecule exhibits 100% sequence identity with thenucleic acid sequence set forth in SEQ ID NO: 7.

In another embodiment, the nucleic acid molecule shares at least 90%sequence identity with the nucleic acid sequence set forth in SEQ ID NO:8. For example, the nucleic acid molecule may share at least 95%sequence identity with the nucleic acid sequence set forth in SEQ ID NO:8, or may share at least 98% sequence identity with the nucleic acidsequence set forth in SEQ ID NO: 8. In a particular embodiment, thenucleic acid molecule has the sequence set forth in SEQ ID NO: 8; thatis, the nucleic acid molecule exhibits 100% sequence identity with thenucleic acid sequence set forth in SEQ ID NO: 8.

Certain polynucleotide and variants thereof are substantially homologousto a portion of a native gene that encodes a desired target polypeptide.Single-stranded nucleic acids derived (e.g., by thermal denaturation)from such polynucleotides and variants are capable of hybridizing undermoderately stringent conditions to a naturally occurring DNA or RNAsequence encoding a native target polypeptide. A polynucleotide thatdetectably hybridizes under moderately stringent conditions may have anucleotide sequence that includes at least 10 consecutive nucleotides,for example, at least 50, at least 100, at least 150, at least 200, atleast 250, and least 300, at least 350, at least 400, at least 450, atleast 500, or more consecutive nucleotides that are complementary to aparticular target polynucleotide. In certain preferred embodiments sucha sequence (or its complement) will be unique to a single particulartarget polypeptide for which interference with expression is desired,and in certain other embodiments the sequence (or its complement) may beshared by two or more related target polypeptides for which interferencewith polypeptide expression is desired.

Sequence specific polynucleotides of the present invention may bedesigned using one or more of several criteria. For example, to design apolynucleotide that has 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, or more, consecutive nucleotides identical to asequence encoding a polypeptide of interest (e.g., a polypeptide havingcolanic acid-degrading activity, such as those described herein), theopen reading frame of the polynucleotide sequence may be scanned forsequences that have one or more of the following characteristics: (1) anA+T/G+C ratio of approximately 1:1 but no greater than 2:1 or 1:2; (2)an AA dinucleotide or a CA dinucleotide at the 5′ end; (3) an internalhairpin loop melting temperature less than 55° C.; (4) a homodimermelting temperature of less than 37° C. (melting temperaturecalculations as described in (3) and (4) can be determined usingcomputer software known to those skilled in the art); (5) a sequence ofat least 10-20 consecutive nucleotides not identified as being presentin any other known polynucleotide sequence (such an evaluation can bereadily determined using computer programs available to a skilledartisan such as BLAST to search publicly available databases).Alternatively, a polynucleotide sequence may be designed and chosenusing a computer software available commercially from various vendors(e.g., OligoEngine™ (Seattle, Wash.); Dharmacon, Inc. (Lafayette,Colo.); Ambion Inc. (Austin, Tex.); and QIAGEN, Inc. (Valencia,Calif.)). See also Elbashir et al., Genes & Development 15:188-200(2000); Elbashir et al., Nature 411:494-98 (2001). The polynucleotidesof interest may then be tested for their ability to encode targetpolypeptides, to hybridize to other polynucleotides of interest, or tointerfere with the expression of the target polypeptide according tomethods known in the art, and the determination of the effectiveness ofa particular polynucleotide based on these tests will be evident to oneof skill in the art.

Persons having ordinary skill in the art will also readily appreciatethat as a result of the degeneracy of the genetic code, many nucleotidesequences may encode a polypeptide as described herein. That is, anamino acid may be encoded by one of several different codons and aperson skilled in the art can readily determine that while oneparticular nucleotide sequence may differ from another (which may bedetermined by alignment methods disclosed herein and known in the art),the sequences may encode polypeptides with identical amino acidsequences. By way of example, the amino acid leucine in a polypeptidemay be encoded by one of six different codons (TTA, TTG, CTT, CTC, CTA,and CTG) as can serine (TCT, TCC, TCA, TCG, AGT, and AGC). Other aminoacids, such as proline, alanine, and valine, for example, may be encodedby any one of four different codons (CCT, CCC, CCA, CCG for proline;GCT, GCC, GCA, GCG for alanine; and GTT, GTC, GTA, GTG for valine). Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention.

The polynucleotide may also comprise a codon optimized sequence; thatis, a nucleotide sequence that has been optimized for a particular hostspecies by replacing any codons having a usage frequency of less thanabout 20%. Nucleotide sequences that have been optimized for expressionin a given host species by elimination of spurious polyadenylationsequences, elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization may be generally referred to in the art as expressionenhanced sequences.

The polynucleotides may also be labeled with reagents that facilitatetheir detection. For example, the agents may be combined withfluorescent labels (e.g., Prober et al., Science 238:336-340 (1987);Albarella et al., EP 144914); chemical labels (e.g., Sheldon et al.,U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No. 4,563,417);and/or modified bases (e.g., Miyoshi et al., EP 0 119 448) (each ofwhich are hereby incorporated by reference in their entirety).

Polynucleotides or fragments thereof of the present invention are alsogenerally capable of specifically hybridizing to other nucleic acidmolecules under certain circumstances. For example, two nucleic acidmolecules are said to be capable of specifically hybridizing to oneanother if the two molecules are capable of forming an anti-parallel,double-stranded nucleic acid structure. A nucleic acid molecule orpolynucleotide is said to be the complement of another nucleic acidmolecule or polynucleotide if they exhibit complete complementarity.Molecules are said to exhibit complete complementarity when everynucleotide of one of the molecules is complementary to a nucleotide ofthe other. Two molecules are said to be minimally complementary if theycan hybridize to one another with sufficient stability to permit them toremain annealed to one another under at least conventionallow-stringency conditions. Similarly, the molecules are said to becomplementary if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another underconventional high-stringency conditions. Conventional stringencyconditions are described elsewhere herein and by Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989) and by Haymes et al., NucleicAcid Hybridization, A Practical Approach, IRL Press, Washington, D.C.(1985), each of which is herein incorporated by reference. Departuresfrom complete complementarity are therefore permissible, as long as suchdepartures do not completely preclude the capacity of the molecules toform a double-stranded structure. Thus, in order for a nucleic acidmolecule to serve as a primer or probe it need only be sufficientlycomplementary in sequence to be able to form a stable double-strandedstructure under the particular solvent and salt concentrations employed.

In a particular embodiment, a polynucleotide of the present inventionwill specifically hybridize to one or more of SEQ ID NO: 7 and SEQ IDNO: 8, or complements thereof, under moderately stringent conditions.

Preparation of Polynucleotides

Polynucleotides, including polynucleotides encoding polypeptides havingcolanic acid-degrading activity, may be prepared using any of a varietyof techniques, which will be useful for the preparation of specificallydesired polynucleotides and for the identification and selection ofdesirable sequences to be used in polynucleotides. For example, apolynucleotide may be amplified from cDNA prepared from a suitablebacteria, cell, or tissue type. Such polynucleotides may be amplifiedvia polymerase chain reaction (PCR). For this approach,sequence-specific primers may be designed based on the sequencesprovided herein and may be purchased or synthesized. An amplifiedportion may be used to isolate a full-length gene, or a desired portionthereof, from a suitable library using well known techniques. Withinsuch techniques, a library (cDNA or genomic) is screened using one ormore polynucleotide probes or primers suitable for amplification.Preferably, a library is size-selected to include larger molecules.Random primed libraries may also be preferred for identifying 5′ andupstream regions of genes. Genomic libraries are preferred for obtainingintrons and extending 5′ sequences. Suitable sequences for apolynucleotide contemplated by the present invention may also beselected from a library of polynucleotide sequences.

For hybridization techniques, a partial sequence may be labeled (e.g.,by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library may then be screened byhybridizing filters containing denatured bacterial colonies (or lawnscontaining phage plaques) with the labeled probe (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 2001). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. Clones may be analyzed to determine the amount of additionalsequence by, for example, PCR using a primer from the partial sequenceand a primer from the vector. Restriction maps and partial sequences maybe generated to identify one or more overlapping clones. A full-lengthcDNA molecule can be generated by ligating suitable fragments, usingwell known techniques.

Alternatively, numerous amplification techniques are known in the artfor obtaining a full-length coding sequence from a partial cDNAsequence. Within such techniques, amplification is generally performedvia PCR. One such technique is known as rapid amplification of cDNA endsor RACE. This technique involves the use of an internal primer and anexternal primer, which hybridizes to a polyA region or vector sequence,to identify sequences that are 5′ and 3′ of a known sequence. Any of avariety of commercially available kits may be used to perform theamplification step. Primers may be designed using, for example, softwarewell known in the art. Primers (or oligonucleotides for other usescontemplated herein, including, for example, probes and antisenseoligonucleotides) are preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31 or 32 nucleotides in length, have a GCcontent of at least 40% and anneal to the target sequence attemperatures of about 54° C. to 72° C. The amplified region may besequenced as described above, and overlapping sequences assembled into acontiguous sequence. Certain oligonucleotides contemplated by thepresent invention may, for some preferred embodiments, have lengths of10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33-35, 35-40, 41-45, 46-50, 56-60, 61-70, 71-80,81-90, or more, nucleotides.

Nucleotide sequences as described herein may be joined to a variety ofother nucleotide sequences using established recombinant DNA techniques.For example, a polynucleotide may be cloned into any of a variety ofcloning vectors, including plasmids, phagemids, lambda phagederivatives, and cosmids. Vectors of particular interest includeexpression vectors, replication vectors, probe generation vectors, andsequencing vectors. In general, a suitable vector contains an origin ofreplication functional in at least one organism, convenient restrictionendonuclease sites, and one or more selectable markers. (See, e.g., PCTInternational Pub. No. WO 01/96584; PCT International Pub. No. WO01/29058; U.S. Pat. No. 6,326,193; U.S. Pub. App. No. 2002/0007051 (eachof which is hereby incorporated by reference herein in its entirety).Other elements will depend upon the desired use, and will be apparent tothose having ordinary skill in the art. For example, the inventioncontemplates the use of polynucleotide sequences in the preparation ofrecombinant nucleic acid constructs including vectors for the expressionof a desired target polypeptide such as a CAE polypeptide; the inventionalso contemplates the generation of transgenic animals and cells (e.g.,cells, cell clones, lines or lineages, or organisms in which expressionof one or more desired polypeptides (e.g., a target polypeptide) isfacilitated). Within certain embodiments, polynucleotides may beformulated so as to permit entry into a cell of a mammal, and expressiontherein. Such formulations are particularly useful for therapeuticpurposes, as described below. Those having ordinary skill in the artwill appreciate that there are many ways to achieve expression of apolynucleotide and/or polypeptide in a target cell, and any suitablemethod may be employed. For example, a polynucleotide may beincorporated into a viral vector using well known techniques (see also,e.g., U.S. Pub. App. No. 2003/0068821 (hereby incorporated by referenceherein in its entirety)). A viral vector may additionally transfer orincorporate a gene for a selectable marker (to aid in the identificationor selection of transduced cells) and/or a targeting moiety, such as agene that encodes a ligand for a receptor on a specific target cell, torender the vector target specific. Targeting may also be accomplishedusing an antibody, by methods known to those having ordinary skill inthe art.

In other embodiments, one or more promoters may be identified, isolatedand/or incorporated into recombinant nucleic acid constructs of thepresent invention, using standard techniques. The present inventionprovides nucleic acid molecules comprising such a promoter sequence orone or more cis- or trans-acting regulatory elements thereof. Suchregulatory elements may enhance expression of a polynucleotide orpolypeptide described herein. A 5′ flanking region may be generatedusing standard techniques, based on the genomic sequence providedherein. If necessary, additional 5′ sequences may be generated usingPCR-based or other standard methods. The 5′ region may be subcloned andsequenced using standard methods. Primer extension and/or RNaseprotection analyses may be used to verify the transcriptional start sitededuced from the cDNA.

To define the boundary of the promoter region, putative promoter insertsof varying sizes may be subcloned into a heterologous expression systemcontaining a suitable reporter gene without a promoter or enhancer.Suitable reporter genes may include genes encoding beta-galactosidase,chloramphenicol acetyl transferase, luciferase, secreted alkalinephosphatase, or the Green Fluorescent Protein (GFP) gene (see, e.g.,Ui-Tei et al., FEBS Lett. 479:79-82 (2000)). Suitable expression systemsare well known and may be prepared using well known techniques orobtained commercially. Internal deletion constructs may be generatedusing unique internal restriction sites or by partial digestion ofnon-unique restriction sites. Constructs may then be transfected intocells that display high levels of polynucleotide and/or polypeptideexpression. In general, the construct with the minimal 5′ flankingregion showing the highest level of expression of reporter gene isidentified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Once a functional promoter is identified, cis- and trans-acting elementsmay be located. Cis-acting sequences may generally be identified basedon homology to previously characterized transcriptional motifs. Pointmutations may then be generated within the identified sequences toevaluate the regulatory role of such sequences. Such mutations may begenerated using site-specific mutagenesis techniques or a PCR-basedstrategy. The altered promoter is then cloned into a reporter geneexpression vector, as described above, and the effect of the mutation onreporter gene expression is evaluated.

Antibodies

One aspect of the present invention concerns antibodies, single-chainantigen binding molecules, or other proteins that specifically bind toone or more of the polypeptides of the present invention and theirhomologues, fusions or fragments. Such antibodies may be used toquantitatively or qualitatively detect the polypeptides of the presentinvention. In general, an antibody or peptide is said to specificallybind to a protein or peptide molecule of the present invention if suchbinding is not competitively inhibited by the presence of non-relatedmolecules.

Polynucleotides that encode all or part of the polypeptide of thepresent invention can be expressed, via recombinant means, to yieldprotein or peptides that can in turn be used to elicit antibodies thatare capable of binding the expressed protein or peptide. Such antibodiesmay be used, for example, in immunoassays for that protein. Suchprotein-encoding molecules, or their fragments may be a fusion molecule(i.e., a part of a larger nucleic acid molecule) such that, uponexpression, a fusion protein is produced. It is understood that any ofthe nucleic acid molecules of the present invention may be expressed,via recombinant means, to yield proteins or peptides encoded by thesenucleic acid molecules.

The antibodies that specifically bind polypeptides and fragments thereofmay be polyclonal or monoclonal and may comprise intact immunoglobulins,or antigen binding portions of immunoglobulins fragments (such as(F(ab′), F(ab′).sub.2), or single-chain immunoglobulins producible, forexample, via recombinant means. It is understood that practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of antibodies (see, for example, Harlow and Lane, In:Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1988), the entirety of which is herein incorporated byreference).

As discussed elsewhere herein, such antibody molecules or theirfragments may be used for diagnostic purposes. Where the antibodies areintended for diagnostic purposes, it may be desirable to derivatizethem, for example with a ligand group (such as biotin) or a detectablemarker group (such as a fluorescent group, a radioisotope or an enzyme).The ability to produce antibodies that bind the protein or peptidemolecules of the present invention permits the identification of mimeticcompounds of those molecules. Generally, mimetic compounds are compoundsthat is not the particular compound of interest, or a fragment of thatcompound, but which nonetheless exhibits an ability to specifically bindto antibodies directed against that compound. In one embodiment, theantibody is a rabbit polyclonal antibody.

Kits

Other aspects of the invention are directed to kits useful in carryingout the processes described herein. In general, the kits for practice ofthe methods of the invention preferably have somewhat different formsdepending on their intended functions

The kits will typically be packaged and include vessels containingreagents, the solution volumes of which will vary based on the amount ofpreparations for which the kit is rated. The vessels will generallyinclude one or more reagents useful in carrying out the processesdescribed herein. In certain embodiments, the kit is a compartmentalizedkit; that is, the kit includes reagents contained in the same orseparate vessels. Examples of vessels include, but are not limited to,small glass containers, plastic containers, or strips of plastic orpaper. These and other similar vessels allow the efficient transfer ofreagents from one compartment to another, or to some other vessel, suchthat the various samples and reagents are not cross-contaminated and theagents or solutions of each container can be added in a quantitativefashion from one compartment to another. Such containers can include acontainer which will accept the test sample, a container which containsthe polypeptide or polynucleotide of the disclosure, a container whichcontains host cells or other materials for producing the polypeptidesdescribed herein (e.g., a vector, virus, or bacteriophage), containerswhich contain chromatography materials (such as one or more of the ionexchange, affinity, and hydrophobic interaction chromatography resinsdescribed above), containers which contain wash reagents (such asphosphate buffered saline, Tris-buffers, and the like), and/orcontainers which contain reagents useful in the detection ofpolysaccharides such as colanic acid (such as those described in theassays detailed below in Example 16). The kit can include sources andconcentrations of the CAE polypeptides described herein. For largerscale applications, the kits will generally include similar reagents andsolutions, but in larger quantity.

For instance, the kit may include a suitable bacterial expression vectorfor cloning the CAE polynucleotides described herein. Alternatively, thekit may include the polynucleotides and/or polypeptides themselves. Thekit may also include cells, such as competent cells, for transformingrecombinant clones into expression vectors. The kit may also includemedia (such as broth) for bacterial expression of the polypeptides ofthe invention. The kits may also include a set of three common alkalinelysis buffers as described in the Qiagen product manual and in Sambrooket al. as Solutions I, II, and III (i.e., 25 mM Tris HCl with 10 mM EDTAat pH 8.0, 1% SDS and 0.2 N NaOH, and 3 M potassium acetate at pH 5.5respectively) and/or a resuspension solution (e.g., 10 mM Tris HCl at pH8.0).

In some embodiments, for example, centrifuge-based spin filters or discfilters can be included. One model spin filter that works for thisapplication is a Millipore Durapore centrifuge filter (MilliporeCorporation, Billerica, Mass.). In addition, or by way of analternative, filters can be included that have a packed steel wool,cellulose or polymer/plastic material in a centrifuge or other filtermechanism (e.g., a disc). Ceramic filters can also be included. Filteraids, such as a diatomaceous earth or similar compound, may also beincluded. For larger scale applications, a tangential-flow filter can beprovided.

In one particular embodiment, the kit includes one or more of thepolypeptides described herein. For example, the polypeptide included inthe kit may be a purified polypeptide comprising an amino acid sequencehaving at least 90%, 98%, 99%, or 100% homology to SEQ ID NO: 1, andconservative amino acid substitutions thereof. In a particularembodiment, the polypeptide may have the amino acid sequence of SEQ IDNO: 1. Additionally or alternatively, the polypeptide included in thekit may be a purified polypeptide comprising an amino acid sequencehaving at least 90%, 98%, 99%, or 100% homology to SEQ ID NO: 2, andconservative amino acid substitutions thereof. In a particularembodiment, the polypeptide may have the amino acid sequence of SEQ IDNO: 2. The polypeptide included in the kit may also include a tag forisolation or purification (e.g., a His-tag or a FLAG®-tag); thus, thepolypeptide included in the kit correspond to SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, or SEQ ID NO: 6. Alternatively, the kit may includereagents and compositions that may be used to form such taggedpolypeptides, along with the polypeptides of SEQ ID NO: 1 or SEQ ID NO:2, and variants thereof.

The kits will also typically include instructions for use. Theinstructions will generally be suitable to enable an end user to carryout the desired preparation or assay. The instructions will generally bein a tangible expression, e.g., describing the reagent concentration forat least one preparation or assay, parameters such as the relativeamount of reagent and sample to be admixed, maintenance or incubationtime periods for reagent/sample admixtures, temperature requirements orpreferences, and the like. The instructions may be printed on the outeror inner packaging of the kit, in a brochure, card, or other paperwithin the kit, and/or on the outer surface of the containers or vesselsincluded in the kit.

Having described the invention in detail, it will be apparent thatmodifications and variations are possible without departing the scope ofthe invention defined in the appended claims. Furthermore, it should beappreciated that all examples in the present disclosure are provided asnon-limiting examples.

EXAMPLES

Colanic acid is present at significant levels in all plasmid DNApreparations, including clinical grade (cGMP) preparations. Colanic Acidcomprises about 25% of the bacterial cell wall of gram negativebacteria. Colanic acid must be removed in order to provide the greatestsafety, especially when mixed with cationic carriers for delivery inanimals and in humans. Removal of colanic acid also increases geneexpression from each plasmid because colanic acid is an inhibitor of RNApolymerase activity. A range of 2.2 to 4.4-fold increased reporter geneexpression (CAT, chloramphenicol acetyltransferase) in the organs ofBalb/c mice post-intravenous (iv) injection of BIV DNA-liposomecomplexes has been observed. Because colanic acid is often extremelylarge and branched-chain, it typically must be degraded in order to beeffectively removed.

With the identification of colanic acid as a primary contaminatingcomponent of plasmid DNA polysaccharides, experiments were performed todevelop methods for removing colanic acid from plasmid DNA. A specificenzyme, referred to hereafter as colanic acid degrading enzyme (CAE),had been reported to be produced by specific lytic bacteriophages(Hughes, K. A. et al. 1998. J. Appl. Microbiol. 85:583-590). The colanicacid degrading enzyme (CAE) had only been partially purified byresearchers. Therefore, in order to develop a method for removal ofcolanic acid using a CAE, the enzyme must be purified, sequenced andexpressed.

A bacteriophage (NST1) was identified that has the ability to lyse theE. coli strain SC12078, a strain that overproduces colanic acid. TheNST1 bacteriophage was isolated and used as a source to isolate apurified CAE. It has been shown that polysaccharide viscosity decreasesafter incubation with a specific polysaccharide degrading enzyme(Sutherland, I. W. 1967. Biochem. J. 104:278-285). Thus, the proteinsamples containing CAE were identified by their ability to affectviscosity of samples containing colanic acid. It was found that thepurified CAE isolated from NST1 had high levels of CAE activity asdemonstrated by its ability to decrease the viscosity of colanic acid.

Once the purified CAE was isolated, as identified by bioassay of theability to decrease sample viscosity, it was subjected to massspectrometry and Edman degradation. Using Edman degradation, 15 aminoacids were identified. By mass spectrometry, 8 additional proteinfragments were sequenced, with each fragment containing between 6 to 16amino acids. Screening of publicly available protein databases,including bacteriophage databases, did not reveal a single match withany of the peptide fragments.

A set of degenerate oligonucleotides was prepared based on the peptidesequences. These oligonucleotides were used to sequence the Colanic AcidDegrading Enzyme from the genomic DNA purified from the NST1bacteriophage. The open reading frame (ORF) of the CAE was determined.The nucleotide CAE ORF was sequenced and the amino acid sequence of CAEdetermined using the universal genetic code. These sequences are shownin FIG. 1. PCR primers made to the beginning and end of the CAE ORF wereused to amplify the ORF sequences from the NST1 bacteriophage genomicDNA by PCR for subsequent cloning into a yeast expression vector.

A naturally occurring colanic acid degrading enzyme (CAE) has beenproduced from bacteriophage that is a newly identified protein;generally, only small amounts are produced, approximately 110 ug from a4.5 L phage+bacterial growth. A rabbit polyclonal antibody to thisprotein has also been produced that is a peptide generated antibody.This antibody is highly active and can be used for any purpose includingWestern blotting, ELISA assay, etc.

In order to produce large-scale amounts of CAE, a recombinant form ofCAE was created for use in further purifying plasmid DNA preparations.Prior attempts to produce full-length recombinant CAE in yeast,baculovirus, and bacteria were generally unsuccessful, believed to bedue to improper protein folding. After examining the predicted structureof the CAE and chymotrypsin digestion of the natural protein, wedetermined that 107 amino acids could be removed from the amino terminus(N-terminus) of the CAE protein without loss of activity. Chymotrypsinwas the only protease that cleaved the natural full-length protein atthis one location, amino acids 106-107. The recombinant CAE protein isnot cleaved by any protease and is extremely stable (>2 years). Weproduced the functional truncated form of CAE in bacteria using theexpression vector pET28a (Invitrogen; see Example 9). The truncatedprotein is produced in Escherichia coli, BL21(DE3) grown at 16° C.overnight and then purified. About 10 mg of CAE recombinant protein from1 L of growth can be produced.

Any plasmid DNA preparation can then be digested with recombinant CAEand further purified. Briefly, plasmid DNA is digested with CAE for 3hours at 37° C. and then at 50° C. for 21 hours. Protein is removed, andthe DNA is first purified by boronic acid chromatography. The plasmidDNA flows through and does not bind the column. Most polysaccharidesexcept for extremely small fragments bind to the column. To remove thesmallest, digested polysaccharides, the DNA suspension is finallypurified by a Macrosep 100 Centrifugal Concentrator unit in the presenceof zwittergent. The zwittergent is generally preferred because colanicacid appears to bind tightly to the plasmid DNA.

For use in the preparation of clinical grade plasmid DNA and to reducecost, recombinant CAE can also be placed on a solid support that can beregenerated and reused multiple times.

By the assays described herein, we find no detectable levels ofpolysaccharides including colanic acid.

Example 1 Preparation of Colanic Acid

Colanic acid was prepared using SC12078 bacteria, a bacterial strainthat is known to overproduce colanic acid. A few colonies of SC12078bacteria were picked from a plate and inoculated into 2 liters of LBbroth with 0.4% glycerol containing chloramphenicol (10 ug/ml). Thebacteria were allowed to grow at 37° C. in a shaker incubator at 230 rpmovernight. The growth was stopped when the cultures reached an opticaldensity (OD) 600 between about 4.5 to about 4.7.

Prior to removing the bacteria by centrifugation, the flasks of bacteriawere briefly shaken to increase the amount of colanic acid released intothe culture medium. The bacteria were pelleted by centrifugation at6,000×g for 15 min at 4° C. The bacterial pellet was discarded and thesupernatant saved and concentrated using an Amicon filter apparatus witha YM30 membrane.

The colanic acid was precipitated from the concentrated supernatant byadding 3 volumes of ice cold ethanol to one volume of supernatant andletting the mixture sit on ice for 15 min. The precipitate was collectedby centrifuging the mixture at 10,000×g for at least 15 min at 0° C., oruntil the supernatant is clear. The precipitate was dissolved in aminimal amount of sterile water and dialyzed overnight against at leastthree changes of water.

The dialyzed solution was lyophilized to dryness, being sure to weighthe tube that the solution was to be dried in before adding the solutionin order to determine the weight of the sample after freeze drying. Oncethe sample was totally dried, water was added to the sample to make a 2%solution of the lyophilized sample.

Solid ammonium sulfate was added to the 2% solution of the lyophilizedsample to achieve a 90% ammonium sulfate saturated solution. The 90%ammonium sulfate saturated solution precipitated the O antigen and thecolanic acid. The precipitated polysaccharides were collected bycentrifugation at 10,000×g for at least 15 min at 0° C., or until thesupernatant was clear. The pelleted precipitate was dissolved in aminimal amount of water, dialyzed overnight against at least threechanges of water, and lyophilized to dryness.

The lyophilate was dissolved in 150 ml of 0.1 M sodium phosphate pH 7.2.The colanic acid was precipitated from the lyophilate solution by adding37.5 ml of hexa-decyl-trimethyl-ammonium bromide (also called cetavlonor cetrimide). The colanic acid precipitate was collected bycentrifugation at 10,000×g for at least 15 min at 0° C., or until thesupernatant was clear.

The pelleted precipitate was dissolved in 100 ml of 1M NaCl. The colanicacid is reprecipitated by adding 3 volumes of ice cold ethanol to the 1MNaCl solution and letting the mixture sit on ice for 15 min. The colanicacid precipitate was collected by centrifuging the mixture at 10,000×gfor at least 15 min at 0° C., or until the supernatant was clear. Thecolanic acid precipitate was dissolved in a minimal amount of sterilewater and dialyzed in a cold room overnight against at least threechanges of water.

The dialyzed solution was lyophilized to dryness, being sure to weighthe tube that the solution was to be dried in before adding the solutionin order to determine the weight of the sample after freeze drying. Oncethe sample was totally dried, the colanic acid was dissolved in aminimal amount of water, aliquoted into sterile tubes, and stored at−25° C.

Example 2 NST1 Phage Production

The NST1 bacteriophage was identified as a good source of CAE by itsability to lyse E. coli strain SC12078, a strain that overproducescolanic acid. The NST1 bacteriophage was isolated and used as a sourceto isolate a purified CAE.

A few colonies of SC12078 bacteria were taken from agar plates andinoculated into two tubes containing 5 ml of LB-glycerol mediacontaining 0.4% chloramphenicol (10 ug/ml). The bacteria were allowed togrow at 37° C. overnight to prepare a phage stock that is. Serialdilutions (1:10², 1:10¹, 1:10⁰, 1:10⁻¹, and 1:10⁻²) are prepared of thephage stock containing five different NST1 phage particle numbers. Thedilutions are based on 1 ul of phage stock stored at 4° C. containing107 phage particles). The overnight growth (200 ul) is mixed with 1 ulof phage stock to make the 10⁷ concentration of phage. Additionaldilutions are made containing 180 ul of overnight bacterial growth mixedwith 20 ul of the next higher concentration of phage. The highestconcentrations containing 107 through 103 particles are discarded.

The lower 5 dilutions were plated by quickly mixing each into 3 ml ofLB+glycerol top agar (agarose at 0.7%, kept at 55° C.). The mixture wasquickly poured onto LB+Chloramphenicol (10 ug/ml) plates. The mixing andpouring is preferably done quickly to avoid solidification of the topagar. The plates were then incubated upside down at 37° C. for 5 hours.After incubation, plates were wrapped with parafilm and stored at 4° C.Plates that do not contain plaques are discarded.

Example 3 Large Scale Production of NST1 Phage Supernatant

The bacterial strain SC12078 (a strain overproducing colanic acid) wasmaintained on LB-containing chloramphenicol (10 ug/ml) agar plates andstored at 4° C. Several plates of NST1 phage, as described in Example 2,were maintained on LB-glycerol top agar (agarose 0.7%), layered on topof LB-chloramphenicol (10 mg/ml) agar plates stored at 4° C. The plateswere not stored for more than one month as NST1 loses viability and itsability to infect bacteria with longer storage time periods.

A few colonies of the SC12078 bacteria were inoculated into four 50 mlsterile tubes, each tube containing 15 ml of LB-glycerol-chloramphenicol(10 ug/ml) media. The colonies were allowed to grow overnight at 37° C.in a shaker incubator.

Three 4 liter flasks were then inoculated with 15 ml of the overnightculture, each flasks containing 1.5 liters ofLB-glycerol-chloramphenicol (10 ug/ml) media. The bacterial colonieswere allowed to grow at 37° C. in a shaker incubator (230 rpm) for about2-4 hours until the solution had an OD600 between 0.12 and 0.67.

Each flask was then inoculated with 30 NST1 phage plugs and the flaskincubated overnight at 37° C. with shaking (230 rpm). The OD600 of theSC12078 cultures inoculated with the NST1 phage that were incubatedovernight was measured and was typically between 4.5 and 4.7. Thesecultures were centrifuged at 4200 rpm at 4° C. for 5 min using large,autoclaved centrifuge bottles. The supernatant, containing the NST1phage, was poured into sterile containers and stored at −80° C., orimmediately purified. The pellets containing the bacterial cells anddebris were discarded.

Example 4 Isolation of the Colanic Acid Degrading Enzyme

Phage supernatant was prepared as described in Example 3. Phenyl methylsulfonyl fluoride (PMSF) was added to the phage supernatant to a finalconcentration of 0.1 mM PMSF to prepare the starting solution and thenstored at 4° C.

The starting solution for CAE purification was centrifuged in thetable-top centrifuged at about 4200 rpm for 20 min at 4° C. Theresulting supernatant was removed and saved and the pellet discarded.Using an Amicon filter apparatus and a YM30 membrane, the supernatantvolume was reduced from 4 liters to a 4 ml sample. The 4 ml sample wasfurther centrifuged in a polycarbonate centrifuge tube at 40,000×g in anSS34 rotor for 60 minutes at 4° C. The sample was dialyzed overnight inthe cold room against at least 3 changes of 10 mM Tris HCl, pH 7.5,containing 0.1 mM PMSF.

A Q SEPHAROSE FAST FLOW™ column (10 cm high, 1.5 cm diameter) wasequilibrated with 10 mM Tris HCl, pH 7.5, 0.1 mM PMSF until the pH ofthe fluid eluting from the column was 7.5. The dialyzed supernatant wasloaded onto the equilibrated column and the column washed with 2 columnvolumes (about 30 ml) of 10 mM Tris HCl, pH 7.5, 0.1 mM PMSF.

The column was eluted using a linear gradient from 10 mM Tris HCl, pH7.5, 0.1 mM PMSF (150 ml) to 200 mM Tris HCl, pH 6.5, 0.1 mM PMSF (150ml) collecting 4 ml fractions (75 fractions total) at a flow rate of 7ml per hour. The fractions collected were tested for colanic aciddegrading activity using a viscometer test, described below in Example5, and those fractions containing CAE activity were pooled.

The pooled CAE active fractions were then concentrated on a disposableAmicon filter by centrifugation. The protein concentration of theresulting concentrate was determined and a sample of the concentrate waselectrophoresed on a gradient polyacrylamide gel (4-12%). Theelectrophoresed sample contained five protein bands.

The protein concentrate was then separated by size on a 120 cm columncontaining TOYOPEARL™ HW-50F resin equilibrated with phosphate bufferedsaline (PBS), pH 7.3-7.4, containing 0.1 mM PMSF. The column eluate wascollected in 1 ml fractions. Each fraction was tested for CAE activityand the active fractions were pooled.

The pooled fractions were concentrated on a disposable AMICON™ filter bycentrifugation. The protein concentration of the concentrate wasdetermined and a sample of the concentrate was electrophoresed on agradient polyacrylamide gel (4-12%). A single protein band was obtainedthat had a molecular weight of about 84,000 Daltons.

The protein band was prepared by standard procedures and submitted formass spectrometric analysis and Edman degradation.

Example 5 Identification of the Partial Amino Acid Sequence of CAEIsolated from Bacteriophage NST1

The CAE protein was purified as described previously. The purified CAEprotein was subjected to mass spectrometry using the Applied BiosystemsProcise Sequencer PROCISE-cLC™ for 17 cycles and Edman degradation.Using Edman degradation, 15 amino acids of the N-terminus wereidentified as set forth below:

ANSYNAYVANGSQTA (SEQ ID NO: 9)

By mass spectrometry, 8 additional protein fragments were sequenced,with each fragment containing between 6 to 16 amino acids as set outbelow:

LLEQGTGEALTDGVLR (SEQ ID NO: 10) VPNSEVSLNALPNVQR (SEQ ID NO: 11)LADYEFTSAPSNSK (SEQ ID NO: 12) YSDLSTLN (SEQ ID NO: 13) QLLFDTAPLA (SEQID: 14) APYQVDDNL (SEQ ID NO: 15) FGAYLPDD (SEQ ID NO: 16) LGTLGG (SEQID NO: 17)

In each of these peptide fragment sequences the amino acid leucine (L)may actually be either leucine (L) or isoleucine (I), the amino acidaspartic acid (D) may actually be aspartic acid (D) or asparagine (N),the amino acid glutamine (Q) may actually be glutamine (Q) or lysine(K), and the amino acid phenylalanine (F) may actually be phenylalanine(F) or oxidized methionine.

Example 6 Cloning the Colanic Acid Degrading Enzyme

A. Preparation of Degenerate Oligonucleotide Primers

Degenerate oligonucleotide primers are prepared using degenerate codonsof the amino acid sequences from the protein fragments of the CAEprotein described above.

B. Preparation of Bacteriophage Genomic DNA

The genomic DNA of the Bacteriophage NST1 was purified using standardDNA purification methods. The degenerate oligonucleotide primers wereused to hybridize with the bacteriophage genomic DNA to identify the CAEgene as described below.

C. Sequencing and Amplification of CAE gene from Genomic DNA

Sequencing of the CAE ORF was performed directly on the NST1bacteriophage genomic DNA starting with the degenerate primers and thenusing primers based on known sequences for subsequent rounds ofsequencing. The CAE ORF was fully sequenced on both strands of the NST1bacteriophage genomic DNA.

Primers made to the beginning and end of the CAE ORF sequences were madeand then used to amplify the sequences of the CAE gene from the NST1bacteriophage genomic DNA using the Polymerase Chain Reaction (PCR)using the Deep Vent DNA Polymerase (New England BioLabs) to avoid errorsduring amplification. The amplified DNA was then electrophoresed and theappropriate band was excised and purified from the gel.

Example 7 Sequence of the Colanic Acid Degrading Enzyme

The nucleotide sequence of the CAE was determined as set out in FIG. 9:

(SEQ ID NO: 7) ATGGCGAACA GCTATAATGC TTACGTGGCG AACGGTTCAC AGACCGCATTCCTCGTCACG 60 TTCGAGCAGC GCGTGTTCAC TGAGATTCAG GTGTACCTCA ACTCCGAACTCCAGACGGAA 120 GGGTACACCT ACAACTCTGT GACCAAACAG ATTATCTTCG ACACCGCCCCGCTCGCCGGG 180 GTGATTGTCC GACTCCAACG CTACACCTCT GAGGTTCTGC TGAACAAGTTTGGCCAAGAC 240 GCTGCCTTCA CCGGGCAGAA CCTTGACGAG AACTTTGAGC AGATTCTGTTCAAGGCTGAG 300 GAAACTCAGG AAGCATGGCT CGCGCCACTT GACCGCGCCG TCCGTGTTCCGAACTCCGAA 360 GTCTCCATCA ACGCATTACC GAACGTCGCT GGCCGCCGCA ACAAGGCACTGGGCTTTGAC 420 AGCAATGGTC AGCCGTTCAT GATTCCTCTG GTCGATATCC CGGACTCCGCGCTGGCGATT 480 GCTCTGGCAA TGGCTGACGG CGGTAAGTGG ATTGGTACTC TCGGCGGGGGCACGTTCCTC 540 GACCGTCAGG ATACCGTCTG CCTGTCCGAG TTCACCAACA ACACTGGGTACGCCTCTGTC 600 GCCGCTGCGG TGCAGGCTTG CTTCGACTAT GCGAAAGCCA ACGGCAAGGTCGTTGACGCT 660 CGCGGCTGGG AAGGTACGGT GGATTCCACT GTGCTGATGG ACGGTATTGAGGTCGTCGGC 720 GGTACGTGGC ACGGCAAGGC TGACATTCGC CTGCTGAACT CCACCTTCCGCAACTTCGTG 780 GCCTCTACTG TCCGTGTCGC CTACTGGGGC GGCGAGGTGC GTATTGCTGACTATGAGTTC 840 ACCAGCGCAC CGAGCAACTC CAAGGTTACG TCTATCCTGT TCCAAGGCAACATCGCCGGG 900 GGCAGCTACG TCATTGAGAA CGGTATCCAC CGCAATGGTA AGTTCGGTATTCTCCAACAG 960 GGTACTGGCG AGGCTATCAC CAACGGCGTT ATCCGTGGCA TCACCATGATGGATATGCAG 1020 GGTGACGGTA TCGAGATGAA CGTAATCAAC AAGCACTATG ATGGTGGCCTGCTGATTGAG 1080 AACATCTTCC TTGAGAACAT CGACGGCACC AACGCGCCTA TCCCACTGTCCAACTGGGGC 1140 ATTGGTATCG GTATCGCTGG TCAAGGCCCG TTCGGTTGGG ATGCTGCTGAGACGCAGTAT 1200 GCGAAGAACG TCACTGTCCG TAACGTCCAT GCTCCGCGTG GTGTGCGTCAGGTCGTCCAC 1260 TTCGAGGTTA CGCGTGACAG CACCTGCGAG AACGTAGTGG CCAACCCTGACCTGTCCGTC 1320 TCCATTGGTA CTGGCCTGAC TGCCGCTGGT GTAATCACGT ACGGCTGCAAGCGCATGACC 1380 ATTGACGGTG TAGTCGGTGA GCCTATGAAC ACCGGAGCAA CCTCTCCGAACGATATTCGT 1440 ATCGTGATGT TGGAGTGGGG TGCGAACCAA GCAGGTGCTG GCGGTACGCCGGGTGCAGCT 1500 TGCCCATCGT TCGACATGAC CGTGCGTAAC GTGCAGACCC GTACCGGGCGCTTCTATGCT 1560 GGTGTCGGCT CCGACGATGA CAACACCAAC ACATATCACC TTGAAAACATTCACTGTTAC 1620 AAGATGACGC TGTTTGGTGT GGCAACTCTG CTGAACATGA CCAACGTGACTGGTGTGGTG 1680 TTCGACGCTG TAGGCGATGA CTCCAGCGGC GGTACGTCCT CCAACGGTCTGTACCCGCGT 1740 AAGAAGACTG TTCTCAACAT GGTGAACGTG AACTTCTACG GGCCGGGCATGACCGAGGGT 1800 GCGCTGTACA GTAAGGCTCG CTACTCGGAT ATCAGCACGC TGAACTCCAACGTGCGTGCT 1860 ATCCCGTACA CCAACATCCA AGGTAACGTG GGTGTCATCC TGTCTCCGGTCAACCGCATG 1920 TACACGCTGC CGAACGCCCT CGCTACCCTT GACGGTAATG AGTTCCCCACCGGGAAAGAG 1980 TTCTGCGAAG GTACTGTGCT GTTCAAGACC GATGGCTCCG GTGGCAACTTCATCGTGACC 2040 CGGTTCGGTG CGTACATCCC GGATGACGGT AACAACTTCA AGGTGCGTGCTGCTGCCGCT 2100 GGCCAGACGT ATCTGGAGCA GAACCTGACT CCGGCTGGTA CTCAGGCTTCCACCTCGTGG 2160 CTGTACCATA AGCCAATCTC TGCTGGTACT CGACTCAATG TTCCGGGTGCCGGGCCGAGC 2220 GGCGGTACGC TCACTGTGAC GGTGGTGCGT GCTCCGTATC AGGTGGACAACAACATCGGA 2280 AACCCGGTAC GCATCGACAT TACCCCGGCC ATTGTGACGG CAATCCCTGCGGGAACGCAG 2340 CTCGCCGCTA CCTACCCGGT GGCTTACATC TAA

The amino acid sequence was determined using the universal genetic codeand is shown in alignment with the nucleotide sequence in FIG. 1. Theamino terminus of the CAE matched the Edman degradation results, exceptthe Edman degradation did not detect the terminal methionine. Inaddition, the molecular weight for the CAE was determined to be 84,354and corresponded to the molecular weight of the protein band sequencedas determined by its position on polyacrylamide gels.

Example 8 Using Colanic Acid Degrading Enzyme in the Purification ofPlasmid DNA

A plasmid DNA sample is tested for the presence of colanic acid. Ifcolanic acid is present in the plasmid DNA sample then the sample ofplasmid DNA is incubated with the recombinant colanic acid degradingenzyme of the present invention. The CAE will digest the colanic acidinto a number of smaller polysaccharides that can be separated from theplasmid DNA by a variety of methods known in the art. The plasmid DNAsample will then be separated from the CAE and further purified asdescribed herein.

Example 9 Construction of Recombinant CAE, Amino Acids 107-790(Underlined) Including 6 Histidines at the N-Terminus, in VectorpET-28a-c(+)

Amino Acids Contained in the Construct: [(Nco1) MGHHHHHH . . . stop(Xho1)] (SEQ ID NO: 4)MGHHHHHHLAPLDRAVRVPNSEVSINALPNVAGRRNKALGFDSNGQPFMIPLVDIPDSALAIALAMADGGKWIGTLGGGTFLDRQDTVCLSEFTNNTGYASVAAAVQACFDYAKANGKVVDARGWEGTVDSTVLMDGIEVVGGTWHGKADIRLLNSTFRNFVASTVRVAYWGGEVRIADYEFTSAPSNSKVTSILFQGNIAGGSYVIENGIHRNGKFGILQQGTGEAITNGVIRGITMMDMQGDGIEMNVINKHYDGGLLIENIFLENIDGTNAPIPLSNWGIGIGIAGQGPFGWDAAETQYAKNVTVRNVHAPRGVRQVVHFEVTRDSTCENVVANPDLSVSIGTGLTAAGVITYGCKRMTIDGVVGEPMNTGATSPNDIRIVMLEWGANQAGAGGTPGAACPSFDMTVRNVQTRTGRFYAGVGSDDDNTNTYHLENIHCYKMTLFGVATLLNMTNVTGVVFDAVGDDSSGGTSSNGLYPRKKTVLNMVNVNFYGPGMTEGALYSKARYSDISTLNSNVRAIPYTNIQGNVGVILSPVNRMYTLPNALATLDGNEFPTGKEFCEGTVLFKTDGSGGNFIVTRFGAYIPDDGNNFKVRAAAAGQTYLEQNLTPAGTQASTSWLYHKPISAGTRLNVPGAGPSGGTLTVTVVRAPYQVDNNIGNPVRIDITPAIVTAIPAGTQLAATYPVAYI stop.

The PCR amplified region of CAE from amino acids 107 to 790, including 8additional amino acids at the N-terminus, was digested with restrictionenzymes Nco1 and Xho1, and ligated into the multiple cloning site ofvector pET-28a-c(+) that was also digested with Nco1 and Xho1. Thisconstruct was transformed and grown in expression host BL21 (DE3) inHyper Broth medium containing 50 mg/liter kanamycin. The large-scaleculture was grown at 37° C. with shaking at 225 rpm until OD₆₀₀ reached0.5 (about 2 to 4 hours). The temperature was changed to 16° C., andgrowth was continued for 30 min. Then IPTG was added to a finalconcentration of 0.015 mM, and growth continued for an additional 20hours. The recombinant CAE is then purified on a Ni-NTA column thatselectively binds the 6 histidines at the N-terminus. The bound proteinis then eluted, concentrated, reconstituted in storage buffer, andstored in the refrigerator at 4° C.

Example 10 Transform Recombinant CAE Clone into Expression Host BL21(DE3)

Thaw on ice one vial of One Shot® BL21 (DE3) cells. Add 10 ng of plasmidDNA, in a volume of 1 to 5 μl, to the cells and mix by tapping gently.Do not mix cells by pipetting. Incubate the vial on ice for 30 minutes.Incubate for exactly 30 seconds in the 42° C. water bath. Do not mix orshake. Remove vial from the 42° C. bath and quickly place on ice. Add250 μl of pre-warmed SOC medium to the vial. Place the vial in amicrocentrifuge rack and secure the vials in the rack with tape. Placethe rack on its side in a shaking incubator, and shake the vial at 37°C. for exactly 1 hour at 225 rpm. Plate 20 to 200 μl each of thetransformation reaction onto two LB plates containing 80 μg/mlKanamycin. Plate two different volumes to ensure well-spaced colonies onat least one plate. The remaining transformation reaction may be storedat +4° C. and plated out the next day, if needed. Invert the plates andincubate at 37° C. overnight.

Example 11 Growing Bacteria Expressing Recombinant CAE

Inoculate a single colony or 30 μl from a glycerol stock into 30 ml LBmedium containing 50 mg/liter kanamycin. Incubate at 37° C. with shakingat 225 rpm overnight. Add 24 ml overnight culture to 2 liters of HyperBroth medium containing 50 mg/liter kanamycin. Grow the culture at 37°C. with shaking at 225 rpm until OD₆₀₀ reaches 0.5 (2˜4 hours). Changetemperature to 16° C., continue shaking for 30 min. Add IPTG to thefinal concentration Of 0.015 mM, continue growing for 20 hours. Harvestcell by centrifugation at 4800×g for 15 min and store the pellet at −80°C.

Example 12 Purification of Recombinant CAE

Make solutions. Add PMSF just before use.

A. 20 mM Tris-HCl pH 8.0, 0.25M NaCl, 10% Glycerol, 10 mM Imidazole, 0.1ml/liter β-mercaptoethanol, 1 mM PMSF.

B. 20 mM Tris-HCl pH 8.0, 0.25M NaCl, 10% Glycerol, 125 mM Imidazole,0.1 ml/liter β-mercaptoethanol, 0.1 mM PMSF.

C. 20 mM Tris-HCl pH 8.0, 0.25M NaCl, 10% Glycerol, 500 mM Imidazole,0.1 ml/liter β-mercaptoethanol, 0.1 mM PMSF.

D. 20 mM Tris-HCl pH 8.0, 0.25M NaCl, 10% Glycerol, 0.1 mM PMSF.

Equilibrate Ni-NTA column (20 ml bed volume, for 2 liter culture) with200 ml of Buffer A. Thaw and suspend the cell pellet (from 2 literculture) in 150 ml of Buffer A. Break cell pastes using MICROFLUIDIZERPROCESSOR™ (Model M-110Y) (according to manufacturer's instructions) andcentrifuge at 40,000×g to obtain clear supernatant. Load supernatant toNi-NTA column and wash with 600 ml of Buffer A, then wash with 80 ml ofBuffer B. Elute bound recombinant CAE with 80 ml of Buffer C. Addelutant to AMICON™ Ultra-4 Centrifugal Filter unit with 30K cutoff. Spinat 2800×g at 4° C. To concentrate elutant. Reconstitute the retentate tothe original sample volume with Buffer D. Repeat this process threetimes. Examine the total protein obtained.

Example 13 Digestion of Plasmid DNA with Recombinant CAE

Purify plasmid DNA from 2.5 liters LB culture using ENDOFREE™ PlasmidGiga Kit (according to manufacture's manufacturer's instructions).Dissolve plasmid DNA in 4 ml of 0.05 M potassium phosphate buffer, pH6.5. Add a suitable amount of recombinant CAE (plasmid DNA: CAE=10:1),incubate at 37° C. for 3 hours. Change temperature to 50° C. andincubate for 21 hours.

Example 14 Boronate Affinity Chromatography

Boronate chromatography has been used to purify samples containing RNA,mononucleotides, oligonucleotides, thymine glycol-containing DNA, andbenzo(a) pyrene: DNA adducts (Schott, H. et al. 1973. Biochemistry12:932-938; Singh, N. and R. C. Wilson. 1999. J. Chromatography840:205-213; Jerkovic, B. et al. 1998. Anal. Biochem. 255:90-94;Pruess-Schwartz, D. et al. 1984. Cancer Res. 44:4104-4110). Boronatechromatography, however, has not been used in the past for successfulpurification of plasmid DNA because no one had identified the presenceof substantial quantities of polysaccharides present in plasmid DNA andthe toxic effects of these polysaccharides during gene therapy.

Commercially available boronic acid affinity resins bind compoundscontaining cis-diol groups. A preferred boronic acid affinity column canbe acquired from Pierce, Rockford, Ill. This boronic acid column has acoupled m-aminophenylboronic acid to polyacrylamide spherical beads at100 mmoles of boronate/ml of gel.

The boronic acid column was equilibrated in 0.2 M ammonium acetate, pH8.8. The plasmid DNA samples were precipitated in ethanol and theprecipitate washed with 70% ethanol. The washed pellet of DNA wasdissolved in 0.2 M ammonium acetate, pH 8.8. The DNA solution was thenloaded onto a boronic acid column at approximately 10 mg DNA per 2 ml ofboronate column material. The column was then washed with 0.2 M ammoniumacetate, pH 8.8. The column wash was collected in fractions and theoptical density (O.D.) of each fraction at 260 nm was measured. Thefractions having the highest O.D. 260 nm were pooled and loaded onto asecond boronic acid column. The second column was washed with 0.2 Mammonium acetate, pH 8.8. Fractions from the column wash were collectedand each of their O.D. 260 nm measured. The fractions having the highestO.D. 260 nm were pooled and the DNA precipitated with ethanol. Theprecipitate was washed with 70% ethanol and resuspended in 10 mM Trisbuffer, pH 8.0. The DNA sample was filter sterilized and stored at −20°C. until it was used.

Plasmid DNA did not bind to the boronate column and flowed through theboronate column with the wash buffer. On the other hand, thepolysaccharide contaminants, RNA, and LPS bound or adsorbed onto theboronate column. Eluting the polysaccharide fractions with 0.1 M formicacid regenerated the boronic acid columns. The boronic acid columns werethen washed and stored in 0.1 M sodium chloride and 0.02% sodium azide.

The purified DNA sample was then subjected to the methods of detectionand quantification for polysaccharides of the present invention. Eachpurified sample was subjected to one or more polysaccharide detectionmethod (i.e., the uronic acid detection method, the fucose detectionmethod, and/or the fluorescence labeling method). The results of thepolysaccharide detection methods consistently demonstrated that the useof boronate chromatography produced DNA samples with polysaccharidecontents that were reduced to undetectable levels. These data areprovided below in Table 2.

Example 15 Macrosep Clean-Up

After Boronic Acid chromatography, pool the fractions having the highestOD260. Precipitate plasmid DNA with 2 volume of 100% cold ethanol, and1/10 volume of 3M sodium acetate, pH 5.2. Incubate at −20° C. (1 hour toO/N). Spin at 13000 rpm for 15 min at 4° C. Wash the pellet twice with 1ml of 70% ethanol, spin at 13000 rpm for 5 min. Air dry and resuspendthe pellet in 14 ml of 10 mM Tris-HCl, pH 8.0, containing 0.1%zwittergent. Incubate at 37° C. for 15 min. Add solution to Macrosep 100Centrifugal Concentrator unit with 300K cutoff (according tomanufacture's instructions). Spin at 3500 rpm for 1 hour at 30° C. toconcentrate plasmid DNA. Reconstitute the retentate to the originalsample volume with 10 mM Tris-HCl, pH 8.0, containing 0.1% zwittergent.Spin at 3500 rpm for 1 hour at 30° C. Repeat this process three times.Reconstitute the retentate to the original sample volume with 10 mMTris-HCl, pH 8.0. Spin at 3500 rpm for 1 hour at 30° C. Repeat thisprocess two times. Precipitate plasmid DNA with 2 volume of 100% coldethanol, and 1/10 volume of 3M sodium acetate, pH 5.2. Incubate at −20°C. (1 hour to O/N). Spin at 13000 rpm for 15 min at 4° C. Wash thepellet twice with 1 ml of 70% ethanol, spin at 13000 rpm for 5 min. Airdry and resuspend the plasmid DNA pellet in sterile water and adjust thefinal concentration to 5 mg/ml.

Example 16 Quantification of Polysaccharides in Plasmid DNA Samples

In order to assure that any method of purification of a plasmid DNAsample successfully removes virtually all of the contaminatingpolysaccharides, a means for assessing polysaccharide contamination ofDNA before and after such purification process was developed.

Three assays were developed, one based on detection of uronic acidlevels (where polysaccharides are known to contain high levels of uronicacid), one based on fucose levels (where fucose is known to make up 22%of colanic acid), and one based on the visual detection offluorescent-labeled polysaccharides in gel-electrophoresed samples.

1. Uronic Acid Assay

E. coli expresses several major classes of polysaccharides, including O-and K-antigen associated polysaccharides, colanic acid, andenterobacterial common antigen (ECA). Colanic acid exists in both highand low molecular weight forms, whereas ECA is typically in a lowmolecular weight form. The O- and K-antigen associated polysaccharideshave variants that are associated with Lipid A and other variants thatare not associated with Lipid A. The Lipid A associated polysaccharidesmay be either covalently linked, or non-covalently linked. The Lipid Aassociated polysaccharides are characteristically low molecular weightvariants. The O- and K-antigen associated polysaccharides that are notassociated with Lipid A exist as high and low molecular weight variants.

Each of these E. coli capsular polysaccharides, particularly thelong-chain and branched polysaccharides found in plasmid DNApreparations, contains uronic acid. For example, colanic acid isapproximately 11 weight % uronic acid. Enterobacterial common antigen(ECA) consists of about 33 weight % uronic acid and the O- and K-antigenassociated polysaccharides have about 25 weight % uronic acid.

The uronic acid content of a plasmid DNA sample was measured usingstandard curves generated with heparin sulfate and glucuronic acid asstandards. Heparin sulfate resembles the polysaccharide contaminantsfrom E. coli, because uronic acid comprises about 25% of the totalweight of heparin sulfate. Heparin sulfate consists of 50% sugars byweight. Half of these sugars are glucosamine and the other half of thesugars are iduronic acid and glucuronic acid. The rest of the heparinsulfate is contributed by modifications of the sugars including sulfatesand acetylamides. Alternatively, glucuronic acid can be used to create astandard curve for the direct measurement of uronic acid.

Standard curves are generated using 0.1 ml of heparin or glucuronic acidstandards containing 0.0, 0.05, 0.1, 0.2, or 0.5 mg of the standard permilliliter of solution. The standard solution (0.1 ml) is placed is aglass test tube with 3 ml of a borate/sulfuric acid solution (i.e.,0.025 M sodium tetraborate 10-hydrate dissolved in sulfuric acid havinga specific gravity of 1.84) and mixed well. A 0.1 ml of a 0.125%solution of carbazole in absolute ethanol is added to the mixture andthe entire mixture is vortexed. The top of each test tube is covered andthe tubes are immersed in boiling water for 10 min. The tubes areallowed to cool and the absorbance of the solution at 530 nm is read ina spectrophotometer. The absorbance values obtained for the standardsare plotted against the concentration of the standards. The uronic acidcontent of plasmid DNA samples can be extrapolated from its absorbancevalue at 530 nm when the DNA sample has undergone the same reaction.

The polysaccharide content of the plasmid DNA sample can then beextrapolated by multiplying the amount of uronic acid by a numberranging from 3.3 to 9.1 (depending on the prevalence of colanic acid,ECA and the O- and K-antigens in the sample). The uronic acid content ofplasmid DNA samples was calculated from standard curves generated withheparin and glucuronic acid standards. The results generated from thetwo standard curves were substantially equivalent.

This method was used to assess the uronic acid content of many plasmidDNA preparations; such DNA preparations included GMP grade DNA preparedby certain companies for use in human drug trials, as well as clinicalgrade DNA. “GMP” is a term used by the U.S. Food and Drug Administrationto designate a compound as having been produced according to regulationsknown as the Good Manufacturing Practice (GMP) regulations. Compoundsproduced by GMP are considered to be safe for use in humans. The resultsof testing various plasmid DNA preparations are presented below in Table2.

TABLE 2 Amount of Uronic Acid DNA Plasmid Preparation/Source Detected(mg/mg DNA) Qiagen Endotoxin Free, Prep A 0.12 Qiagen Endotoxin Free,Prep B 0.18 GMP Grade DNA, Company 1 0.11 Clinical Grade DNA, Company 10.11 GMP Grade DNA, Company 2 0.11 GMP Grade DNA, Company 3, Prep A 0.20GMP Grade DNA, Company 3, Prep B 0.22 GMP Grade DNA, Company 4, Prep A0.22 GMP Grade DNA, Company 4, Prep A 0.25 Plasmid DNA preparedaccording to 0.00 present disclosure

2. Fucose Assay

Since gram-negative bacteria are known to consist of approximately 25%colanic acid, plasmid DNA preparations were also subjected to an assayfor colanic acid. The colanic acid assay was based on the amount offucose present per mg of DNA. Colanic acid consists of 22% fucose in theratio of 2:2:1:1:3 (fucose:galactose:glucose:uronic acid:othermodifications), whereas the other polysaccharide contaminants do notcontain fucose, or only small amounts of fucose. Thus, the fucose assayallowed for identification of the amount of colanic acid contaminationin a purified plasmid DNA preparation.

For example, a plasmid DNA preparation containing about 0.7 mgpolysaccharide per mg of DNA, as estimated by the uronic acid assay, had0.14 mg of fucose per mg of DNA or about 0.64 mg of colanic acid. It wasgenerally found that the primary polysaccharide contaminant in plasmidDNA preparations was colanic acid.

Since colanic acid was present in high levels in even clinical gradeplasmid DNA, it is necessary to assure that any method of purificationof a plasmid DNA sample successfully removes virtually all of thecontaminating colanic acid. Thus, a method for assessing colanic acidcontamination of DNA before and after such purification process wasdeveloped.

The basic procedures for assay of fucose content in samples can be foundin a paper by Morris (Morris, J. B. 1982. Anal. Biochem. 121:129-134).Detailed descriptions of the solution preparation and storage conditionsfor solutions and samples for this assay have also been published(Passonneau, J. V. and O. H. Lowry. 1974. In: Methods of EnzymaticAnalysis, U. H. Bergmeyer (ed.), 2nd edition, Academic Press: New York,volume 4, pp. 2059-2072).

DNA samples to be analyzed (450 ug) are transferred to 3 ml vials andlyophilized. To each sample, 200 ul of 5.5 M trifluoroacetic acid isadded and the reaction vials are sealed with a TEFLON™-lined cap.Hydrolysis is accomplished by heating the samples for 4 hours at 100° C.After cooling to room temperature, the trifluoroacetic acid is removedwith a stream of argon gas under a fume hood. The remaining residue isthen redissolved in 212 ul of sterile water. Only 200 ul of theresulting sample, which corresponds to 425 ul of the initial DNA sample,is used in the fucose assay.

Using the method described by Morris (1982. Anal. Biochem. 121:129-134),200 ul of the samples and standards are pipetted into 1.5 ml microcentrifuge tubes. The standards used are fucose solutions ranging inconcentration from 0 ug fucose to 200 ug fucose (0, 1, 12.5, 25, 50, 100and 200 ug fucose).

The tubes are placed on ice and 50 ul of a 1 mg/ml fucose dehydrogenasesolution is added, followed by 50 ul of 200 uM NAD+. The tubes are mixedand incubated for 3 hours at 4° C. To stop the enzymatic reaction, 50 ulof 1 N NaOH is added to each tube, mixed, and incubated for 10 minutesat 60° C. The tubes are then cooled on ice and the samples are mixedwith 50 ul of 1 M hydrochloric to neutralize the samples.

From each sample, a 50 ul aliquot is removed and transferred to a fresh1.5 ml micro centrifuge tube. To each sample, 250 ul of cycling reagent(200 mM Tris pH 8.4; 50 mM ammonium acetate; 0.5 mM ADP; 100 mM lactate;5 mM alpha-ketoglutarate; 20 units/ml lactate dehydrogenase; 20 units/mlglutamate dehydrogenase) is added, the solution is mixed, and incubatedfor 1 hour at room temperature. Heating each tube for 2 minutes inboiling water stops the enzymatic reaction of the cycling reagent.

The tubes are cooled on ice and then 250 ul of pyruvate reagent (800 mMimidazole buffer, pH 6.2; 0.45 mM NADH; 0.06 units/ml lactatedehydrogenase) is added and the tubes mixed. The tubes are then warmedfor 1 to 2 minutes at room temperature in a water bath before placementin an incubator at 30° C. for 20 minutes. The pyruvate reaction isstopped by adding 200 ul of 1.5 M HCl to each sample and mixing thesolution.

The contents of each tube are then transferred to a 15 ml capped tubeand 2.5 ml of 6 N NaOH is added and mixed. The tubes are then incubatedfor 10 minutes at 60° C. After cooling the samples to room temperature,4 ml of sterile water is added to each tube, the tubes inverted and thensubjected to fluorescence measurement.

A part of each sample (300 ul) is aliquoted into a well of a 96-wellmicrotiter plate. Three to five wells are filled with sterile water andused as blanks. The fluorometer is set using a 360 nm excitation filterand a 465 nm emission filter. The fluorescence of the standards andsamples is read and the fluorescence of the blanks subtracted out. Thefluorescence readings of the standards are graphed as a standard curveand the amount of fucose in the plasmid DNA samples is determined byinterpolation from the standard curve.

Using this method, the fucose levels of plasmid DNA samples weredetermined and the concentration of colanic acid levels calculated.Colanic acid was consistently found to be the primary contaminant inplasmid DNA from a variety of sources, even GMP grade plasmid DNA.

3. Fluorescent Detection of Gel Electrophoresed DNA Samples

A visual method developed for the detection of polysaccharides inplasmid DNA samples involved labeling of the samples with a substancecapable of selectively labeling polysaccharides in a plasmid DNA sample.One such substance is DTAF, (4,6-dichlorotriazinyl)aminofluorescein(Molecular Probes, Eugene, Oreg.). DTAF specifically labels allpolysaccharides whether or not they contain uronic acid. Thisfluorescence probe reacts with hydroxyl groups found in polysaccharidesor carbohydrates and is therefore a probe with application beyond theassay method using uronic acid detection.

DTAF does not label DNA, since DNA does not have free hydroxyl groupsavailable. All of the available hydroxyl groups in DNA arephosphorylated. This specificity makes DTAF the preferred label fordistinguishing DNA from its polysaccharide contaminants. Although DTAFwas used in the method of the present invention, one of skill wouldunderstand that any fluorescence label that provides for specificity oflabeling between polysaccharides and DNA would be useful in the methodof the present invention.

DNA and polysaccharide can be visualized in parallel samples run on onegel. DNA is pretreated with ethidium bromide (EtBr) before adding thesamples to the gel. Polysaccharides are labeled with DTAF. A plasmid DNAsample can be run in two lanes on one gel with the sample in one lanestained with EtBr and the sample in the other lane stained with DTAF;thereby, allowing one to visualize the polysaccharide and DNA content ofa plasmid DNA sample.

DNA and polysaccharide standards (40 ul of a 2 mg/ml solution) wereprecipitated by the addition of 10 ul of 3M sodium acetate, pH 5.2,followed by 200 ul of cold ethanol. The samples were incubated for 30minutes at −20° C., then centrifuged 4 minutes at 10,000 rpm in anEppendorf microfuge. The precipitates were then suspended in 10 ulsodium acetate plus 200 ul ethanol and recentrifuged. The precipitateswere then resuspended in 200 ul ethanol, recentrifuged and dissolved in50 ul 0.1 M sodium carbonate, pH 10.5.

A fresh DTAF suspension is prepared by suspending 60 mg/ml DTAF incarbonate buffer. Since not all of the DTAF will go into solution, theresulting suspension is vortexed before its addition to each sample. A 5ul sample of the fresh DTAF suspension (which has been kept dark andcold) is added to the dissolved sample at timed intervals of 0 minutes,45 minutes, and 90 minutes. Upon each addition of the DTAF suspension,the reaction mixture is vortexed and placed at room temperature in thedark. The reaction is terminated after 2.5 hours by precipitating eachsample with the addition of 10 ul sodium acetate and 325 ul ethanol.Incubating these samples for 45 minutes at −20° C. encourages theprecipitation. The samples are then centrifuged and the precipitateswashed 3 times with 25 ul sodium acetate and 500 ul ethanol. The samplesare finally washed with 500 ul ethanol and then dried for 20 minutes atroom temperature in the dark.

The washed samples are dissolved in 40 ul Tris Acetate EDTA (TAE) bufferand 2.5 ul to 20 ul of the sample are applied to the gel. DNA samplesthat were not reacted with DTAF were added to other lanes in the gel inthe presence of EtBr. Lambda DNA-Hind III Digest and PhiX174 DNA-HaeIIIDigest are run as gel markers.

The gel is a 1% agarose Tris Acetate EDTA gel, pH 8.3. Neither the gelnor the running buffer contain ethidium bromide. The gel iselectrophoresed for 45 minutes at 90 volts. It is important to note thatthe sample buffer must be free of bromophenol blue, which will quenchthe DTAF fluorescence, except in the Lambda Hind III marker lane.

FIG. 2 shows a gel where several different DNA plasmid samples weretested using this gel electrophoretic method for polysaccharidevisualization and quantification. LPS (Sigma Chemical Company, St.Louis, Mo.) and “detoxified” LPS, where the fatty acid portions of theLipid A have been removed, (Sigma Chemical Company, St. Louis, Mo.) wereadded as controls in lanes 10 and 8 respectively of the gel. Lanes 1-4and 12 illustrate the DTAF staining of plasmid DNA samples for which theuronic acid content is given in Table 1. Lane 5, 7, 9, 11 and 13 have nosample loaded. Lane 6 illustrates the DTAF staining of a Qiagenendotoxin free DNA sample (currently considered the gold standard forpurified plasmid DNA).

Lanes 14 through 20 of the gel illustrated in FIG. 2 show the results ofEtBr staining of different DNA samples. Lanes 14-17 are the same DNAsamples stained with EtBr that are stained with DTAF in Lanes 1-4. Lane18 is the DNA sample shown in Lane 12 stained with EtBr. Lane 19 is theEtBr stain of the Qiagen endotoxin-free DNA sample, shown in Lane 6stained with DTAF. Lane 20 is a mixture of high and low DNA molecularweight markers labeled with EtBr.

The results showed that the polysaccharides and the DNA migrate atdifferent locations on the gel and that DTAF only labeledpolysaccharides contained in the DNA samples and ethidium bromide onlylabeled DNA. All of the DNA samples, including the Qiagen endotoxin-freeDNA, had detectable levels of polysaccharides. LPS and detoxified LPSalso contained detectable levels of polysaccharides. Therefore, althoughthe “detoxified” LPS (Sigma Chemical Company, St. Louis, Mo.) hassupposedly had all Lipid A removed, there were still significant levelsof polysaccharides detected in Lane 8 which contained the detoxified LPSsample.

These data are provided below in Table 3.

TABLE 3 Polysaccharide DNA Plasmid Method of Detected PreparationDetection (mg/mg DNA) Boronate-purified uronic acid assay undetectable,≦0.05 plasmid DNA Boronate-purified DTAF/EtBr gels undetectable byplasmid DNA visualization

4. Toxicity of Polysaccharides Found in Plasmid DNA Samples

To determine whether the levels of polysaccharide detected in plasmidDNA samples were clinically significant, acute toxicity studies of thesamples were performed in animals. Balb/c mice, six weeks old, wereinjected intravenously with DNA-liposome complexes, where the liposomeswere prepared according to published procedures (Nancy Smyth Templeton,et al. July 1997. Nature Biotechnology 15:647-652). Twenty mice in eachgroup were injected and followed for one week post-injection. Theresults are shown in Table 4.

Results showed that the intravenous injection of 100 ul of DNAcontaining 0.4 mg polysaccharide per mg DNA caused all of the mice todie within 18 hours post-injection. In contrast, the injection of 50 ulof the same DNA did not cause any of the animals to dies within a weekafter injection. Similar results were obtained using DNA samples fromvarious sources.

Plasmid DNA containing levels of about 0.26 mg polysaccharide per mg DNAwere found to reduce gene expression when 50 ug DNA were injected intoimmune compromised transgenic mice once a week for three months. Whenplasmid DNA preparations contained undetectable levels of polysaccharideper mg DNA (<0.03 mg), there were no adverse effects in the animals.

TABLE 4 # Dead # Dead Mice 1 Wk # Mice Mice 18 hr Post- Source of DNA[DNA] Injected Post-Injection Injection Clean DNA* 100 μg 20 0 0 CleanDNA*  50 μg 20 0 0 Qiagen Endo-Free 100 μg 20 20 NA Qiagen Endo-Free  50μg 20 0 0 GMP, Company #1 100 μg 20 20 NA GMP, Company #1  50 μg 20 0 0GMP, Company #2 100 μg 20 20 NA GMP, Company #2  50 μg 20 0 0 CleanDNA + 0.4 mg 100 μg 20 20 NA polysaccharide/mg DNA Clean DNA + 0.4 mg 50 μg 20 0 0 polysaccharide/mg DNA Liposomes**  0 20 0 0 *Clean DNAcontains no detectable polysaccharide as measured by the uronic acidassay or staining with DTAF. **The quantity of liposomes injected wereequivalent to the quantity injected with 100 μl of DNA.

We also performed additional in vivo studies in mice to test our DNApurification procedure using the truncated CAE recombinant protein.These studies were performed in normal mice (Balb/c), and in SCID micewith or without pancreatic tumors. SCID mice are more sensitive tocolanic acid and die at iv injections containing 40 ug of commerciallyproduced plasmid DNA complexed to liposomes and other cationic carriers,whereas Balb/c mice die at levels just above 50 ug of plasmid DNA. Inthe tables show in FIGS. 12-14, we showed that a total of 120 micesurvived high doses of DNA-BIV liposomal complexes post-iv injections,purified according to the processes of the invention.

5. Assay for Detecting CAE Using BCA Reagent

Principle: Colanic Acid Enzyme (6×His-CAE) and other carbohydrasesincrease the reducing ends when degrading their substrates. Theminiaturized highly sensitive bicinchoninic acid (BCA) reducing valueassay, presented in this protocol detects reducing ends of sugars. Thisassay can be used for the detection of all carbohydrases degrading anypolysaccharide; enzymes with either an exo- or endo-type of mechanisms.However caution should be exercised to reduce background absorbancecaused by culture medium, proteins and substrates.

Material and Equipment:

Colanic Acid (CA): 2 μg/μl in 0.05 M potassium phosphate buffer pH 6.5

6×His-CAE: 0.5 μg/μl in 0.05 M potassium phosphate buffer pH 6.5

Bovine Serum Albumin (BSA): 0.5 μg/μl in 0.05 M potassium phosphatebuffer pH 6.5

Buffer: 0.05 M potassium phosphate buffer pH 6.5

MICRO BCA™ Protein Assay Kit (Pierce Product #23235)

Optical 96-Well Reaction plate (Applied Biosystems, Part #4306737)

96 Well, Flat bottom Non Tissue Culture tested, Nonsterile PVC FlexiblePlate (Falcon#353912) or Fisherbrand Flat Bottom 96 Well plate, clear,PS, non-sterile (Cat#12565501)

Multichannel pipet

96 Well Plate Reader

37° C. and 50° C. Incubator

Centrifuge with rotor for spinning 96 well plate

Brief Procedure: Briefly, this assay uses a 96 well format, with eachwell contains a mixture of 2 μg of CAE (TEST) or BSA (BLANK), 100 μg CA,in a total volume of 110 μl of Buffer which is incubated at 37° C. for 3hours, and later at 50° C. for 21 hours. Subsequently 100 μl of eachreaction mix is transferred to a new 96 well plate, to which 100 μl offreshly prepared BCA reagent is added and allowed to incubate at 37° C.for 2 hours, cooled to RT for 15 min, and readings at 550 nm are takenusing a multiplate reader.

Detailed Procedure: In an Optical 96-Well Reaction plate (AppliedBiosystems), Aliquot Blank total volume is 110 μl (i.e. Buffer+CA+BSA)in triplicates, i.e. add the following in the same sequence, 56 μlBuffer+50 μl of 2 μg/μl CA+4 μl of 0.5 μg/μl BSA.

Aliquot Test total volume is 110 μl (i.e. Buffer+CA+6×His CAE) intriplicates, i.e. add the following in the sequence, 56 μl Buffer+50 μlof 2 μg/μl CA+4 μl of 0.5 μg/μl 6×His-CAE.

Seal all the wells using the strip caps provided. Spin the plate at 1500rpm at RT for 1 min. Remove caps from the wells, and mix by gentlypipeting up and down three times using Multichannel pipet. Reseal thewells using the caps and incubate plate in 37° C. incubator for 3 hours.

Shift plate to 50° C. incubator and incubate overnight for a total of 21hours.

Spin plate at 1500 rpm at RT for 1 min.

Use a multichannel pipet to aliquot 100 μl of each well sample to a new96 well plate (Falcon or Fisherbrand).

Prepare BCA reagent by mixing the three reagents provided in the MICROBCA™ kit, i.e., MA+MB+MC in a ration of 0.5:0.48:0.02 using enough to besufficient for all the wells. Aliquot 100 μl of this freshly preparedBCA reagent to each of the wells using a multichannel pipet, mixinggently by pipeting three times up and down. Put lid on 96 well plate,and incubate plate at 37° C. incubator for 2 hours. Let plate cool atroom temp for 15 mins, and take reading at 550 nm using multiplatereader.

To get the value of CAE activity subtract values of Blank from the Test.Note: You can take readings at other wavelengths if the Multiplatereader does not allow you to take at 550 nm, For example at 544 nm, 570nm, 595 nm. But readings are best near to the 550 nm.

References: P. J. A. Meeuwsen, J.-P. Vincken, G. Beldman and A. G. J.Voragen, J. A Universal Assay for screening expression libraries forcarbohydrases. Biosci. Bioeng. 89 (2000), pp. 107-109; Verhoef R,Beldman G, Schols H A, Siika-aho M, Rättö M, Buchert J, Voragen A G.Characterisation of a 1,4-beta-fucoside hydrolase degrading colanicacid. Carbohydr Res. 2005 Aug. 15; 340(11):1780-1788.

6A. Viscosity Assay for the Detection of CAE in Plasmid DNA Preparations

Principle: Colanic Acid (CA) is viscous, and there is a reduction of itsviscosity when it is catalytically degraded by Colanic Acid Enzyme(6×His-CAE). This drop in viscosity can be accurately measured using aviscometer and can be used to calculate activity of CAE by comparing itto the values obtained using a Control reaction i.e., containing CA butin the absence of the enzyme.

Material and Equipment:

Colanic Acid (CA) 1.2 mg/ml in 0.05 M Potassium Phosphate Buffer pH 6.5

Colanic Acid Enzyme (6×His-CAE) 1.0 μg/μl in 0.05 M Potassium PhosphateBuffer pH 6.5

Buffer: 0.05 M Potassium Phosphate Buffer pH 6.5

Water Bath at 37° C.

Brookfield DV-I+ Viscometer

Brookfield CPE 40 spindle

Brief Procedure:

600 μg CA was mixed with 1 μg CAE in the presence of 0.05 M PotPhosphate Buffer pH 6.5, in a total volume of 500 μl. Mix an incubate at37° C. using a water bath. Let cool to RT for 10 min, and take readingsusing a viscometer. This is compared to a Control reaction in theabsence of CAE.

Detailed Procedure:

For CONTROL, in a 1.5 ml eppendorf tube, aliquot 0.5 ml of 1.2 mg/mlCA+50 μl 0.05 M Pot Phosphate Buffer pH 6.5.

For TEST, in a 1.5 ml eppendorf tube, aliquot 0.5 ml of 1.2 mg/ml CA+1μl of 6×His-CAE (1.0 μg/μl)+49 μl 0.05 M Pot Phosphate Buffer pH 6.5.

Mix by gently pipeting up and down, and incubate at 37° C. for 1 hourusing a water bath. Remove tubes from water bath and let cool to RT for10 min.

Measure Viscosity of Test and Control using a Brookfield DV+Viscometer,set at 100 rpm, for 30 seconds using 0.5 ml of the respective reactionmix aliquoted into a CPE 40 spindle.

Note down Viscosity values as CentiPoise (cP) for both Test and Control,and calculate CAE activity using cP values as follows: The calculationis based on the fact that water which has no viscosity has a cP value of1.00. If CAE has 100% activity it would reduce the viscosity of CA to acP value of 1.00. Hence for eg If the CONTROL has a cP value of 1.57, wewould subtract 1.00 from it to get a value of 1.57−1.00=0.57 whichrepresents 100% activity (that is if the cP value drops by 0.57 in thetest it would have 100% CAE activity). If the TEST has a cP value of1.05, we would similarly subtract this cP value from that of theControl. i.e. 1.57−1.05=0.52, this figure would be used to calculateactual CAE activity as follows: 0.57 is 100% activity, hence 0.52 is howmuch % activity? 0.52 multiplied by 100 and divided by 0.57=91.22% CAEactivity.

6B. Viscometry Testing for Identification of CAE Activity

A bioassay measuring changes in the viscosity of colanic acid sampleswas used to detect CAE. A decrease the viscosity of colanic acid sampleswas indicative of CAE activity. The viscosity of the colanic acidsamples, before and after incubation with an enzyme fraction, wasmeasured using a Wells-Brookfield Cone Plate viscometer with a CPE-40cone. This viscometer provided the most sensitive measurement of changesin viscosity in small volumes of about 500 ul. Viscometer accuracy wasmonitored by measuring the viscosity of a mineral oil standard andcomparing the reading with the known viscosity for mineral oil.

The CAE assay utilized a 500 ul sample of a 1.5% colanic acid solution.Diluted enzyme samples or controls samples, between 50 to 100 ul, wasadded to the 1.5% colanic acid solution. Controls for the enzyme assayswere prepared by adding the diluted enzyme samples to buffer and byadding non-proteinaceous samples to the 1.5% colanic acid solution. Eachtest sample (enzyme samples or control samples) was incubated for onehour at 37° C. After the one hour incubation, the test samples wereallowed to adjust to room temperature for 10 minutes.

The viscosity of all test samples was taken at room temperature for 30seconds at 100 rpm. All test samples having CAE activity, isolated andpurified as described above, demonstrated a significant decrease inviscosity, as compared to the control samples that showed no change inviscosity. The CAE test samples decreased the viscosity of the 1.5%colanic acid solution from 1.51 to 0.99, where 0.99 was the lowestviscometry reading obtainable using the Wells-Brookfield Cone Plateviscometer and was equal to the viscometry reading of the buffercontrols.

Therefore, the present invention provides both a method of detection andquantification of polysaccharides in plasmid DNA samples and a methodfor removing polysaccharide from plasmid DNA samples to levels ofpolysaccharides below levels that produce clinically significanttoxicity.

It is to be understood that this invention is capable of otherembodiments and of being practiced and carried out in various ways.Also, it is to be understood that the phraseology and terminologyemployed herein is for the purposes of description and should not beregarded as limiting.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and/or methods and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A process for digesting colanic acid in abiological material, the process comprising contacting the biologicalmaterial under conditions that cause colonic acid digestion with apolypeptide having at least 95% sequence identity to SEQ ID NO: 2 thatdigests colanic acid.
 2. The process of claim 1, wherein the polypeptideis a recombinant polypeptide.
 3. The process of claim 1, wherein thepolypeptide comprises an amino acid sequence having at least 98%sequence identity to SEQ ID NO: 2, and conservative amino acidsubstitutions thereof.
 4. The process of claim 1, wherein thepolypeptide is SEQ ID NO: 2.